Aryloxypropylamines as chemosensitizing agents in the treatment of cancer

ABSTRACT

A method of treating a subject having cancer, particularly a multidrug resistance cancer, which comprises administering to the subject at least one chemotherapeutic agent and at least one 3-aryloxy-3-phenylpropylamine and pharmaceutical compositions and kits for implementing the method.

FIELD AND BACKGROUND OF THE INVENTION

[0001] The present invention relates generally to the field of oncology,and to methods and pharmaceutical compositions for enhancing theactivity of a cancer chemotherapeutic agent. More particularly, thepresent invention concerns the use of a 3-aryloxy-3-phenylpropylaminesuch as fluoxetine [(N-methyl3-(p-trifluoromethylphenoxy)-3-phenylpropylamine] as a chemosensitizerfor enhancing the cytotoxicity of a chemotherapeutic agent, especiallyin drug-resistant tumors and more particularly in the case of MultidrugResistance (MDR). Methods and compositions are provided for thetreatment of cancers such as, but not limited to, leukemia, lymphoma,carcinoma and sarcoma (including glioma) using a3-aryloxy-3-phenylpropylamine, fluoxetine in particular, as achemosensitizer.

[0002] Many of the most prevalent forms of human cancer resist effectivechemotherapeutic intervention. Some tumor populations, especiallyadrenal, colon, jejunal, kidney and liver carcinomas, appear to havedrug-resistant cells at the outset of treatment (Barrows, L. R.,“Antineoplastic and Immunoactive Drugs”, Chapter 75, pp 1236-1262, in:Remington: The Science and Practice of Pharmacy, Mack Publishing Co.Easton, Pa., 1995). In other cases, a resistance-conferring geneticchange occurs during treatment; the resistant daughter cells thenproliferate in the environment of the drug. Whatever the cause,resistance often terminates the usefulness of an antineoplastic drug.

[0003] Clinical studies suggest that a common form of multidrugresistance in human -cancers results from the expression of the MDR1gene that encodes P-glycoprotein. This glycoprotein functions as aplasma membrane, energy-dependent, multidrug efflux pump that reducesthe intracellular concentration of cytotoxic drugs. This mechanism ofresistance may account for de novo resistance in common tumors, such ascolon cancer and renal cancer, and for acquired resistance, as observedin common hematologic tumors such as acute nonlymphocytic leukemia andmalignant lymphomas. Although this type of drug resistance may becommon, it is by no means the only mechanism by which cells become drugresistant. MDR is effected via an extrusion mechanism (Tan B,Piwnica-Worms D, Ratner L., Multidrug resistance transporters andmodulation. Curr. Opin. Oncol, 2000 Sep;12(5):450-8). The influx ofchemotherapeutic drugs into cells is mainly by passive diffusion acrossthe cell membrane, driven by the drug's electrochemical-potentialgradient. In multidrug resistance cells there are energy-dependantextrusion channels that actively pump the drug out of the cells,reducing its intracellular concentration below lethal threshold. Thefirst pump identified was named Pgp (for P-glycoprotein), the second wasnamed MRP (for Multidrug Resistant associate Protein) and several morehave been identified in recent years (Tan et al. 2000, ibid.). All ofthem are naturally occurring proteins, and their physiological roles areassumed to involve detoxification of cells. In multidrug resistancecells they are present, for reasons yet unknown, in a significantlyhigher number of copies than in other non-multidrug resistance cells.Hereinafter, these proteins acting as extrusion pumps or channels inmultidrug resistance cells are referred to, interchangeably, as “MDRpumps”, “MDR extrusion pumps”, “extrusion pumps”, “MDR channels”, “MDRextrusion channels” and “extrusion channels”.

[0004] Chemical modification of cancer treatment involves the use ofagents or maneuvers that are not cytotoxic in themselves, but modify thehost or tumor so as to enhance anticancer therapy. Such agents arecalled chemosensitizers. Pilot studies using chemosensitizers indicatethat these agents may reverse resistance in a subset of patients. Thesesame preliminary studies also indicate that drug resistance ismultifactorial, because not all drug-resistant patients haveP-glycoprotein-positive tumor cells and only a few patients appear tobenefit from the use of current chemosensitizers.

[0005] Chemosensitization research has centered on agents that reverseor modulate multidrug resistance in solid tumors by modulating theactivity of the MDR extrusion pumps. Chemosensitizers known to modulatethe function of MDR extrusion pumps, e.g., P-glycoprotein, include:calcium channel blockers (Verapamil, indicated for the treatment ofhypertension), calmodulin inhibitors (trifluoperazine), indole alkaloids(reserpine), quinolines (quinine), lysosomotropic agents (chloroquine),steroids, (progesterone), triparanol analogs (tamoxifen), detergents(cremophor EL), and cyclic peptide antibiotics (cyclosporines, indicatedto prevent host vs. graft disease) (DeVita, V. T., et al., in Cancer,Principles & Practice of Oncology, 4th ed., J. B. Lippincott Co.,Philadelphia, Pa., pp 2661-2664, 1993; Sonneveld P, Wiemer E. Inhibitorsof multidrug resistance., Curr Opin Oncol 1997 Nov;9(6):543-8).

[0006] A review of studies where chemosensitizing agents were usedconcluded the following: (i) cardiovascular side effects associated withcontinuous, high-dose intravenous Verapamil therapy are significant anddose-limiting; (ii) dose-limiting toxicities of the chemosensitizers,trifluoperazine and tamoxifen, was attributed to the inherent toxicityof the chemosensitizer and not due to enhanced chemotherapy toxicity;(iii) studies using high doses of Cyclosporin A as a chemosensitizerfound hyperbilirubinemia as a side effect; and (iv) further research isclearly needed to develop less toxic and more efficaciouschemosensitizers to be used clinically (DeVita et al., 1993, ibid.).

[0007] For example, while Verapamil is effective in hypertensiontreatment at the 2-4 μM range, for MDR reversal it requires the doserange of 10-15 μM, while at 6 μM it is already in the toxic domain.

[0008] Tumors that are considered drug-sensitive at diagnosis butacquire an MDR phenotype at relapse, pose an especially difficultclinical problem. At diagnosis, only a minority of tumor cells mayexpress proteins such as P-glycoprotein, which act as extrusion pumpsand treatment with chemotherapy provides a selection advantage for thefew cells that are, for example, P-glycoprotein positive early in thecourse of disease. Another possibility is that natural-product-derivedchemotherapy actually induces the expression of MDR1, leading toP-glycoprotein-positive tumors or other MDR pump-positive tumors atrelapse. Using chemosensitizers early in the course of disease mayprevent the emergence of MDR by eliminating the few cells that areMDR-pump positive at the beginning. In vitro studies have shown thatselection of drug-resistant cells by combining Verapamil and Doxorubicindoes prevent the emergence of P-glycoprotein, but that an alternativedrug resistance mechanism develops, which is secondary to alteredtopoisomerase II function (Dalton, W. S., Proc. Am. Assoc. Cancer Res.31:520, 1990).

[0009] More efficacious and less toxic chemosensitizers are urgentlyneeded to improve the outcome of chemotherapy. Clinical utility of achemosensitizer depends upon its ability to enhance the cytotoxicity ofa chemotherapeutic drug and also on its low toxicity in vivo. Thepresent inventors have addressed these problems and provide herein a newclass of chemosensitizers that permit new approaches in cancertreatment.

[0010] 3-Aryloxy-3-phenylpropylamines and their use to treat depressionare described in, for example, U.S. Pat. Nos. 4,018,895 and 6,258,853.Fluoxetine [(N-methyl 3-(p-trifluoromethylphenoxy)-3-phenylpropylamine],known better by its commercial name Prozac, is a well-known approveddrug, indicated for psychiatric treatments (Cookson J, Duffett R.,Fluoxetine: therapeutic and undesirable effects. Hosp Med 1998Aug;59(8):622-6). It is known to be an SSRI (Selective SerotoninReuptake Inhibition) agent, and this activity is considered to berelated to its mechanism of action in its capacity as a psychiatric drug(Cookson et al., 1998, ibid.).

[0011] WO 94/18961, WO 92/11035, and U.S. Pat. Nos. 5,798,339 and5,859,065, which are incorporated by reference as if fully set forthherein, disclose methods of treating cancer using histamine antagonistsfollowed by chemotherapy. Specifically, the methods described in thesedocuments are directed at increasing the cytotoxicity and inhibiting theadverse side effects of a chemotherapeutic agent used in chemotherapy,and are effected by administering the chemotherapeutic agent followingthe administration of a histamine antagonist. WO 94/18961 teaches inthis respect that histamine antagonists inhibit normal cellproliferation, while promoting malignant cell proliferation and furtherteaches DPPE analogs as preferred compounds that act as histamineantagonists that affect cell proliferation as described. WO 94/18961recites fluoxetine amongst other psychiatric agents which can also actas histamine antagonists, but further states that this group ofcompounds, at their effective histamine antagonizing concentration,cause adverse side effects, such as cardiac arrhythmia.

[0012] In the experiments described in WO 94/18961, doses equivalent to20-40 mg/M² of fluoxetine were employed. These experiments show that atthis dose range, fluoxetine promotes the proliferation of fibrosarcomacells and inhibits the proliferation of concavalin A-stimulated normallymphocytes. It will be appreciated in this regard that the known,acceptable safety limit of fluoxetine is 80 mg/M², while the safe,substantially side effect-free, daily dose range of fluoxetine is below10-15 mg/M².

[0013] According to WO 94/18961 and WO 92/11035, the histamineantagonists are administered prior to the administration of thechemotherapeutic agent. WO 92/11035 clearly indicates that theantagonist compound is administered about 15 to about 90 minutes,preferably, about 30 to about 60 minutes, prior to the administration ofthe chemotherapeutic agent, in order to permit the antagonist to inhibitthe binding of intracellular histamine to its receptor in normal cellsand thereby, in effect, inhibit the proliferation of normal cells andhence provide chemoprotection to such cells.

[0014] Although WO 94/18961 teaches the use of fluoxetine in a method oftreating cancer, as a compound which is administered in combination witha chemotherapeutic drug, WO 94/18961 does not teach the use offluoxetine as a chemosensitizer used for enhancing the cytotoxic effectof a chemotherapeutic agent in the treatment of multidrug resistancecancer cells.

[0015] Rather, WO 94/18961 teaches that fluoxetine acts as a compoundthat inhibits proliferation of normal cells while promoting theproliferation of cancer cells, when administered prior to thechemotherapeutic drug, at a dose which causes adverse side effects. BothWO 94/18961 and WO 92/11035 do not address the issue of multidrugresistance cancer cells and fail to indicate the use of the methodsdisclosed therein for treating multidrug resistance cancer. In fact, themethods taught in these publications, employ, as is described in theExamples section thereof, cancer cells that are known to be susceptibleto chemotherapeutic treatment, such as S-10 and fibrosarcoma cells.Furthermore, U.S. Pat. Nos. 5,798,339 and 5,859,065, which correspond tothese international publications, specifically recite methods oftreating cancer cells which are susceptible to chemotherapy treatment.Based on the mechanism of action of histamine antagonists disclosed inthese publications, one would be reluctant from administering fluoxetineat its histamine antagonizing dose to a cancer patient not only becauseof its associated side effects, but also because it is said and shown toenhance the proliferation of cancer cells at these concentrations.

[0016] Hence, 3-Aryloxy-3-phenylpropylamines in general and fluoxetinein particular have not hitherto been indicated as chemosensitizers forthe treatment of multidrug resistance cancer.

SUMMARY OF THE INVENTION

[0017] While reducing the present invention to practice it wasunexpectedly found that fluoxetine, a member of the3-aryloxy-3-phenylpropylamines family of compounds, induces asignificant enhancement of the cytotoxic effect of conventionalchemotherapeutic drugs, acting via totally different cytotoxicmechanisms, at a dose range well below fluoxetine's toxicity limits andfurther well below fluoxetine's side effect-free limit. Such anenhancement of the cytotoxic effect of chemotherapeutic drugs isparticularly advantageous in the treatment of multidrug resistancecancer cells.

[0018] Hence, the present invention provides methods, pharmaceuticalcompositions and kits for chemosensitization using a3-aryloxy-3-phenylpropylamine as a chemosensitizing agent.

[0019] As used herein, the term “chemosensitization” means an increaseor an enhancement of the measured cytotoxicity of a chemotherapeuticagent on multidrug resistance cells in the presence of achemosensitizing agent, as is compared to the level of cytotoxicityexerted by the chemotherapeutic agent in the absence of thechemosensitizing agent.

[0020] As shown herein, 3-aryloxy-3-phenylpropylamines act aschemosensitizing agents, rendering multidrug resistance cancer cellsmore sensitive to chemotherapy.

[0021] Hence, in one aspect, the present invention provides a method oftreating a subject suspected of having, or having, a multidrugresistance (MDR) cancer. The method comprises administering to thesubject a chemotherapeutically effective amount of a chemotherapeuticagent and a chemosensitizing effective amount of a3-aryloxy-3-phenylpropylamine. The cancer may be leukemia, lymphoma,carcinoma or sarcoma.

[0022] In a preferred embodiment, the chemotherapeutic agent and the3-aryloxy-3-phenylpropylamine are administered substantially at the sametime.

[0023] In another preferred embodiment, the chemosensitizing dose of the3-aryloxy-3-phenylpropylamine is within its safety range, and moreover,it is within its side effect-free range, so as to avoid adverse sideeffects. Preferably the range used is between about 0.1 mg/M² and about10 mg/M².

[0024] As used herein, the term about indicates ±20%.

[0025] The phrases “side effect-free range” and “side effect-free limit”indicate a dose range and a maximal dose, respectively, that are withinthe safety limit and are further below the minimal dose thatsubstantially induces adverse side effects. In other words, thesephrases indicate safe, substantially free of side effects, range andlimit, respectively. As is demonstrated hereinabove, fluoxetine, forexample, induces side effects even at concentrations that are within itssafety limit. As the safety limit of fluoxetine is a daily dose of 80mg/M², fluoxetine induces side effects such as cardiac arrhythmia at adose range of 20-40 mg/M². Hence, the side effect-free range offluoxetine includes daily doses that are below its side effect-freelimit, namely, below 15 mg/M² preferably, below 10 mg/M².

[0026] In another aspect of the invention,3-aryloxy-3-phenylpropylamines may be used as topical chemosensitizers.For example, Table 2 below indicates that 5-fluorouracil is usedtopically in the treatment of premalignant skin lesions. The inventorsof the present invention envision the use of3-aryloxy-3-phenylpropylamines to enhance the cytotoxicity ofchemotherapeutic agents formulated applicable for topicaladministration.

[0027] A method for selecting a chemotherapeutic agent for which3-aryloxy-3-phenylpropylamine acts as a chemosensitizer is a furtheraspect of the present invention. The method comprises (i) assayingcytotoxicity of a candidate chemotherapeutic agent in the presence andin the absence and optionally at different concentrations of a3-aryloxy-3-phenylpropylamine; and (ii) selecting a candidatechemotherapeutic agent as a chemotherapeutic agent for which3-aryloxy-3-phenylpropylamine is a chemosensitizer when the cytotoxicityof the candidate agent is greater in the presence of3-aryloxy-3-phenylpropylamine than in the absence of3-aryloxy-3-phenylpropylamine. A presently preferred in vitro assay isthe MTT cytotoxicity assay which is described in the examples section.An exemplary in vivo assay is described in, for example, U.S. Pat. No.5,776,925, which is incorporated herein by reference.

[0028] Preferably, the method according to this aspect of the presentinvention is performed with multidrug resistant cells, with a dose of3-aryloxy-3-phenylpropylamine that ranges between about 1 μM and about15 μM, preferably, about 1 μM and about 12 μM and/or while administeringthe 3-aryloxy-3-phenylpropylamine and the candidate chemotherapeuticagent substantially at the same time.

[0029] According to further aspects of the present invention, there areprovided pharmaceutical compositions and pharmaceutical kits.

[0030] In one embodiment, the pharmaceutical composition of theinvention comprises a 3-aryloxy-3-phenylpropylamine as achemosensitizing agent and a chemotherapeutic agent.

[0031] The pharmaceutical composition is preferably packaged in apackaging material and is identified in print in or on the packagingmaterial for use in the treatment of multidrug resistance cancer.

[0032] In another embodiment, the pharmaceutical composition of theinvention comprises a 3-aryloxy-3-phenylpropylamine and thepharmaceutical composition is packaged in a packaging material and isidentified in print in or on the packaging material for use inchemosensitization.

[0033] The pharmaceutical kit of the present invention comprises a3-aryloxy-3-phenylpropylamine as a chemosensitizing agent and achemotherapeutic agent, which are individually packaged in the kit.

[0034] A 3-aryloxy-3-phenylpropylamine used as a chemosensitizer inaccordance with the teachings of the present invention is preferably ofthe formula:

[0035] wherein each R′ is independently hydrogen or methyl;

[0036] R is naphthyl or

[0037] R″ and R′″ are halo, trifluoromethyl, C₁-C₄ alkyl, C₁-C₃ alkoxyor C₃-C₄ alkenyl; and

[0038] n and m are 0, 1 or 2; and acid addition salts thereof formedwith pharmaceutically acceptable acids.

[0039] In the above formula when R is naphthyl, it can be eitheralpha-naphthyl or beta-naphthyl. R″ and R′″ when they are halo, C₁-C₄alkyl, C₁-C₃ alkyloxy or C₃-C₄ alkenyl represent, illustratively, thefollowing atoms or groups: fluoro, chloro, bromo, iodo, methyl, ethyl,isopropyl, n-propyl, n-butyl, isobutyl, sec-butyl, t-butyl, methoxy,ethoxy, n-propoxy, isopropoxy, allyl, methallyl, crotyl and the like. Rthus can represent o, m and p-trifluoromethylphenyl, o, m andp-chlorophenyl, o, m and p-bromophenyl, o, m and p-fluorophenyl, o, mand p-tolyl, xylyl including all position isomers, o, m and p-anisyl, o,m and p-allylphenyl, o, m and p-methylallylphenyl, o, m andp-phenetolyl(ethoxyphenyl), 2,4-dichlorophenyl, 3,5-difluorophenyl,2-methoxy-4-chlorophenyl, 2-methyl-4-chlorophenyl,2-ethyl-4-bromophenyl, 2,4,6-trimethylphenyl,2-fluoro-4-trifluoromethylphenyl, 2,4,6-trichlorophenyl, 2,4,5-trichlorophenyl and the like.

[0040] Also included within the scope of the present invention are thepharmaceutically acceptable salts of the amine bases represented by theabove formula formed with non-toxic acids. These acid addition saltsinclude salts derived from inorganic acids such as: hydrochloric acid,nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydriodicacid, nitrous acid, phosphorous acid and the like, as well as salts ofnon-toxic organic acids including aliphatic mono and dicarboxylates,phenyl-substituted alkanoates, hydroxy alkanoates and alkanedioates,aromatic acids, aliphatic and aromatic sulfonic acids etc. Suchpharmaceutically-acceptable salts thus include: sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, nitrate, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, fluorodide, acetate,propionate, decanoate, caprylate, acrylate, formate, isobutyrate,caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, terephthalate,benzenesulfonates, toluenesulfonate, chlorobenzenesulfonate,xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate,citrate, lactate, beta-hydroxybutyrate, glycollate, malate, tartrate,methanesulfonate, propanesulfonates, naphthalene-1-sulfonate,naphthalene-2-sulfonate, mandelate and the like salts.

[0041] Compounds illustrative of the scope of this invention include thefollowing:

[0042] 3-(p-isopropoxyphenoxy)-3-phenylpropylamine methanesulfonate;

[0043] N,N-dimethyl 3-(3′,4′-dimethoxyphenoxy)-3-phenylpropylaminep-hydroxybenzoate;

[0044] N,N-dimethyl 3-(alpha-naphthoxy)-3-phenylpropylamine bromide;

[0045] N,N-dimethyl 3-(beta-naphthoxy)-3-phenyl-1-methylpropylamineiodide;

[0046] 3-(2′-methyl-4′,5′-dichlorophenoxy)-3-phenylpropylamine nitrate;

[0047] 3-(p-t-butylphenoxy)-3-phenylpropylamine glutarate;

[0048] N-methyl 3-(2′-chloro-p-tolyloxy)-3-phenyl-1-methylpropylaminelactate;

[0049] 3-(2′,4′-dichlorophenoxy)-3-phenyl-2-methylpropylamine citrate;

[0050] N,N-dimethyl 3-(m-anisyloxy)-3-phenyl-]-methylpropylaminemaleate;

[0051] N-methyl 3-(p-tolyloxy)-3-phenylpropylamine sulfate;

[0052] N,N-dimethyl 3-(2′,4′-difluorophenoxy)-3-phenylpropylamine2,4-dinitrobenzoate;

[0053] 3-(o-ethylphenoxy)-3-phenylpropylamine dihydrogen phosphate;

[0054]N-methyl-(2′-chloro-4′-isopropylphenoxy)-3-phenyl-2-methylpropylaminemaleate;

[0055] N,N-dimethyl 3-(2′-alkyl-4′-fluorophenoxy)-3-phenylpropylaminesuccinate;

[0056] N,N-dimethyl 3-(o-isopropoxyphenoxy)-3-phenyl-propylamine phenylacetate;

[0057] N,N-dimethyl 3-(o-)bromophenoxy)-3-phenyl-propylaminebeta-phenylpropionate;

[0058] N-methyl 3-(p-iodophenoxy)-3-phenyl-propylamine propiolate;

[0059] N-methyl 3-(3-n-propylphenoxy)-3-phenyl-propylamine decanoate;and preferably, N-methyl3-(p-trifluoromethylphenoxy)-3-phenylpropylamine.

[0060] The present invention successfully addresses the shortcomings ofthe presently known configurations by identifying new chemosensitizerswhich efficiently act at concentrations well below their toxicity andwhich are of particular efficacy in chemosensitizing multi drugresistant (MDR) cancer cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

[0062] In the drawings:

[0063]FIG. 1 is a bar graph demonstrating the increase in death of C6cells and, separately, PANC-1 cells (% from untreated control) as afunction of treatment media, at 24 hours post administration. CS—15 μMfluoxetine alone, CT—0.1 μg/ml Doxorubicin alone, CT+CS—combination ofthe two. Each bar is an average of 32-64 repeats, and the error barsrepresent the standard deviations. The star (*) indicates statisticalsignificance P<0.001 (two-tails student t-test) compared to thetreatment with the chemotherapeutic drug alone.

[0064]FIG. 2 is a bar graph demonstrating the increase in death of C6cells and, separately, PANC-1 cells (% from untreated control) as afunction of treatment media, at 24 hours post administration. CS—15 μMfluoxetine alone, CT—30 μg/ml Mitomycin C alone, CT+CS—combination ofthe two. Each bar is an average of 32-64 repeats, and the error barsrepresent the standard deviations. The star (*) indicates statisticalsignificance P<0.001 (two-tails student t-test) compared to thetreatment with the chemotherapeutic drug alone.

[0065]FIG. 3 is a dose response curve for the effects of fluoxetine onthe survival of PANC-1 cells treated with 0.1 μg/ml Doxorubicin or,separately, 0.3 μg/ml Vinblastine, 48 hours post administration. Thepoints are experimental, each an average of 32-64 repeats (sd levelswhich are similar to those in FIGS. 1 and 2, are not shown in order toreduce symbol crowding). The solid curves are non-theoretical, drawn toemphasize the trends in the data.

[0066]FIG. 4 is a bar graph demonstrating LD₅₀ doses of fluoxetineeffect in potentiating tumor treatment by the chemotherapeutic drugsDoxorubicin and, separately, Vinblastine, for five different cell lines.Data was taken from analysis of dose response curves similar to thoseshown in FIG. 3, for each of the cell lines, obtained under the samedrug species, drug dose and treatment period listed in the legend toFIG. 3.

[0067]FIG. 5 is a scheme illustrating the MDR mechanism. Thechemotherapeutic drug, denoted CT, usually gains entry into the cell byself-diffusion, this influx driven by the electrochemical gradient ofthe drug across the cell membrane. The intracellular drug concentrationis reduced below lethal threshold, by ATP-dependant extrusion throughthe MDR pumps embedded in the cell membrane.

[0068]FIG. 6 demonstrates the efflux of intracellular Doxorubicin (DOX)from C6 cells, under unidirectional flux conditions. The efflux isexpressed as f(t), the cumulative quantity of DOX that diffused out ofthe cells at time=t, normalized to the total intracellular DOXconcentration at time=0. The points are the experimental data, opensquares—for cells loaded with 0.1 μg/ml DOX and open circles for cellsloaded with 0.1 μg/ml DOX together with 15 μM fluoxetine. The solidcurves are non-theoretical, drawn to emphasize the trends in the data.

[0069]FIG. 7 is a bar graph demonstrating the chemosensitizer-inducedintracellular accumulation of Rhodamine-123 in C-26 cells, expressed inRhodamine-123 intracellular fluorescence. Each bar is an average ofthree independent experiments and the standard deviation (sd) isexpressed by the error bar.

[0070]FIGS. 8a-c presents confocal microscopy images demonstrating theintracellular accumulation of Rhodamine-123 in monolayers of C-26 cellsincubated with 5 μM Rhodamine-123 alone (FIG. 8a), 5 μM Rhodamine-123and 15 μM Verapamil (FIG. 8b) and 5 μM Rhodamine-123 and 15 μMfluoxetine (FIG. 8c).

[0071]FIG. 9 presents comparative plots demonstrating an increase in asolid tumor volume with time, in each of the tested groups in in vivostudies of a solid tumor model. Each point in the plots represents anexperimental measurement and is an average of 5 animals. The error barsrepresent the SEM and the curves are non-theoretical, indicating thetrends in the data. The results presented in the left-hand side plotswere obtained with Mitomycin C (MMC) as the chemotherapeutic drug andthe results presented in the right-hand side plots were obtained withDoxorubicin (DOX) as the chemotherapeutic drug.

[0072]FIG. 10 presents the survival data in each of the tested groups inthe solid tumor model of FIG. 9.

[0073]FIGS. 11a and 11 b are bar graphs demonstrating the increase inlung weight (FIG. 11a) and the number of tumor metastasis (FIG. 11b) indifferent mice groups injected with B16F10 cells. Each bar is an averageof all the animals in the group and the sd is represented by the errorbars.

[0074]FIG. 12 presents the survival data obtained in each of the testedgroups in the lung metathesis model of FIGS. 11a-b.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0075] The present invention is of methods and pharmaceuticalcompositions which can be used in chemosensitization. Specifically, thepresent invention can be used to render cancer cells and, in particularmultidrug resistance cancer cells, more sensitive to chemotherapeuticagents, hence increase the cytotoxic effect of such agents on cells.

[0076] The principles and operation of a method and pharmaceuticalcomposition according to the present invention may be better understoodwith reference to the drawings and accompanying descriptions.

[0077] Before explaining at least one embodiment of the invention indetail, it is to be understood that the invention is not limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways. Also,it is to be understood that the phraseology and terminology employedherein is for the purpose of description and should not be regarded aslimiting.

[0078] The present invention results from the discovery that3-aryloxy-3-phenylpropylamines act as efficient chemosensitizers onmultidrug resistance cells at non-toxic concentrations.Chemosensitization using a 3-aryloxy-3-phenylpropylamine refers to anenhancement of cytotoxicity on the part of a chemotherapeutic agent whenthat agent is administered to multidrug resistance cells in conjunctionwith administering a 3-aryloxy-3-phenylpropylamine.

[0079] Hence, the invention relates to a novel treatment for effectingtumor (both solid and non-solid) chemotherapy, based on the combinationof at least one chemotherapeutic drug that are used in, for example,standard therapy protocols in the clinic such as, but not limited to,Doxorubicin, Vinblastine and Mitomycin C; and at least one3-aryloxy-3-phenylpropylamine, preferably fluoxetine (also known asProzac), a drug approved and widely used for psychiatric situations suchas depression.

[0080] It is shown herein that a combined treatment of at least onechemotherapeutic drug and at least one 3-aryloxy-3-phenylpropylamineleads to significant increases in efficacy of the cytotoxic drugs, up to5-fold for a single dose, and at doses well below safety limits and sideeffect-free limits known for 3-aryloxy-3-phenylpropylamines. Moreover,the novel treatment is especially effective in tumors that are resistantto the chemotherapeutic drugs, i.e., multidrug resistance tumors.

[0081] It will be appreciated in this respect that other multidrugresistance reversal agents, such as Verapamil and Cyclosporin A, are notused in the clinic due to their toxicity at the required dose levels.

[0082] In addition, 3-aryloxy-3-phenylpropylamines, such as fluoxetine,can be administered orally, which is easier on the patient, and itspresently indicated effect of mood improvement will also be beneficialto the cancer patient.

[0083] According to the present invention the chemotherapeutic agent maybe, for example, one of the following: an alkylating agent such as anitrogen mustard, an ethylenimine and a methylmelamine, an alkylsulfonate, a nitrosourea, and a triazene; an antimetabolite such as afolic acid analog, a pyrimidine analog, and a purine analog; a naturalproduct such as a vinca alkaloid, an epipodophyllotoxin, an antibiotic,an enzyme, a taxane, and a biological response modifier; miscellaneousagents such as a platinum coordination complex, an anthracenedione, ananthracycline, a substituted urea, a methyl hydrazine derivative, or anadrenocortical suppressant; or a hormone or an antagonist such as anadrenocorticosteroid, a progestin, an estrogen, an antiestrogen, anandrogen, an antiandrogen, or a gonadotropin-releasing hormone analog.Specific examples of alkylating agents, antimetabolites, naturalproducts, miscellaneous agents, hormones and antagonists, and the typesof cancer for which these classes of chemotherapeutic agents areindicated are provided in Table 2. Preferably, the chemotherapeuticagent is a nitrogen mustard, an epipodophyllotoxin, an antibiotic, or aplatinum coordination complex. A more preferred chemotherapeutic agentis Bleomycin, Vinblastine, Doxorubicin, Paclitaxel, etoposide, 4-OHcyclophosphamide, or cisplatinum.

[0084] Presently preferred chemotherapeutic agents are Doxorubicin,Mitomycin C and/or Vinblastine, which are the chemotherapeutic drugsemployed in the in vitro and/or in vivo experiments described in theExamples section that follows, yet the use of other chemotherapeuticdrugs in context of the present invention is also applicable.

[0085] As is well known in the art, Vinblastine, Mitomycin C andDoxorubicin are cytotoxic drugs toward which many tumors exhibit drugresistance and therefore serve as representative examples for thechemosensitization effect of 3-aryloxy-3-phenylpropylamine.

[0086] Furthermore, as is delineated in Table I below, these drugsdiffer from one other by their chemical structure, mechanism of actionand the location of their cellular targets. For example, Vinblastineacts in the cytosol, via depolymerization of microtubules, Mitomycin Cacts in the nucleus via DNA alkylation and Doxorubicin acts in both thecytosol and the nucleus, as well as in the cell membrane, havingdifferent effects in each of these locations. TABLE 1 Intracellularlocation of Cytotoxic drug drug target Mechanisms of action VinblastineThe cytosol Depolymerization of Microtubules Mitomycin C The nucleus DNAalkylation Doxorubicin The nucleus DNA intercalation, blocking synthesisof DNA and RNA The cytosol DNA strands scission by affectingtopoisomerase II The cell Altering membrane fluidity and ion membranetransport Generation of semiquinone free radicals and oxygen radicals

[0087] Hence, as these drugs act via different pathways, the presentinventors envision that 3-aryloxy-3-phenylpropylamines may be used aschemosensitizers for enhancing the cytotoxicity of a variety ofchemotherapeutic agents having different mechanisms of action.

[0088] A listing of currently available chemotherapeutic agentsaccording to class, and including diseases for which the agents arepresently indicated, is provided as Table 2 below. Each of theseexemplary chemotherapeutic agents can be used in the context of thepresent invention. TABLE 2 Chemotherapeutic Agents Useful in NeoplasticDisease¹ Class Type of Agent Name Disease² Alkylating NitrogenMechlorethamine Hodgkin's disease, non-Hodgkin's Agents Mustards (HN₂)lymphomas Cyclophosphamide Acute and chronic lymphocytic Ifosfamideleukemias, Hodgkin's disease, non-Hodgkin's lymphomas, multiple myeloma,neuroblastoma, breast, ovary, lung, Wilms' tumor, cervix, testis,soft-tissue sarcomas

lphalan Multiple myeloma, breast, ovary

lorambucil Chronic lymphocytic leukemia, primary macroglobulinemia,Hodgkin's disease, non- Hodgkin's lymphomas Estramustine ProstateEthylenimines Hexamethylmelamine Ovary and Methylmelamines ThiotepaBladder, breast, ovary Alkyl Busulfan Chronic granulocytic leukemiaSulfonates Nitrosoureas Carmustine Hodgkin's disease, non-Hodgkin'slymphomas, primary brain tumors, multiple myeloma, malignant melanomaLomustine Hodgkin's disease, non-Hodgkin's lymphomas, primary braintumors, small-cell lung Semustine Primary brain tumors, stomach, colonStreptozocin Malignant pancreatic insulinoma, malignant carcinoidTriazenes Dacarbazine Malignant melanoma, Hodgkin's Procarbazinedisease, soft-tissue sarcomas Aziridine Antimetabolites Folic AcidMethotrexate lymphocytic leukemia, Analogs Trimetrexate choriocarcinoma,mycosis fungoides, breast, head and neck, lung, osteogenic sarcomaPyrimidine Fluorouracil Breast, colon, stomach, pancreas, AnalogsFloxuridine ovary, head and neck, urinary bladder, premalignant skinlesions (topical) Cytarabine Acute granulocytic and acute Purine AnalogsAzacitidine lymphocytic leukemias and Related Mercaptopurinelymphocytic, acute Inhibitors granulocytic, and chronic granulocyticleukemias Thioguanine Acute granulocytic, acute lymphocytic, and chronicgranulocytic leukemias Pentostatin Hairy cell leukemia, mycosisfungoides, chronic lymphocytic leukemia Fludarabine Chronic lymphocyticleukemia, Hodgkin's and non-Hodgkin's lymphomas, mycosis fungoidesNatural Vinca Alkaloids Vinblastine (VLB) Hodgkin's disease,non-Hodgkin's Products lymphomas, breast, testis Vincristine Acutelymphocytic leukemia, neuroblastoma, Wilms' tumor, rhabdomyosarcoma,Hodgkin's disease, non-Hodgkin's lymphomas, small-cell lung VindesineVinca-resistant acute lymphocytic leukemia, chronic myelocytic leukemia,melanoma, lymphomas, breast Epipodophyl- Etoposide Testis, small-celllung and other Lotoxins Teniposide lung, breast, Hodgkin's disease,non-Hodgkin's lymphomas, acute granulocytic leukemia, Kaposi's sarcomaAntibiotics Dactinomycin Choriocarcinoma, Wilms' tumor,rhabdomyosarcoma, testis, Kaposi's sarcoma Daunorubicin Acutegranulocytic and acute lymphocytic leukemias Doxorubicin Soft-tissue,osteogenic, and 4′- other sarcomas; Hodgkin's Deoxydoxorubicin disease,non-Hodgkin's lymphomas, acute leukemias, breast, genitourinary,thyroid, lung, stomach, neuroblastoma Bleomycin Testis, head and neck,skin, esophagus, lung, and genitourinary tract; Hodgkin's disease, non-Hodgkin's lymphomas Plicamycin Testis, malignant hypercalcemia MitomycinStomach, cervix, colon, breast, pancreas, bladder, head and neck EnzymesAsparaginase Acute lymphocytic leukemia Taxanes Docetaxel Breast,ovarian Paclitaxel Biological Interferon Alfa Hairy cell leukemia,Kaposi's Response sarcoma, melanoma, carcinoid, Modifiers cell, ovary,bladder, non-Hodgkin's lymphomas, mycosis fungoides, multiple myeloma,chronic granulocytic leukemia Tumor Necrosis Investigational FactorTumor- Investigational Infiltrating Lymphocytes Miscellaneous PlatinumCisplatin Testis, ovary, bladder, head and Agents CoordinationCarboplatin neck, lung, thyroid, cervix, Complexes endometrium,neuroblastoma, osteogenic sarcoma Anthracenedione Mitoxantrone Acutegranulocytic leukemia, breast Substituted Hydroxyurea Chronicgranulocytic leukemia, Urea polycythemia vera, essential thrombocytosis,malignant melanoma Methyl Procarbazine Hodgkin's disease HydrazineDerivative Adrenocortical Mitotane Adrenal cortex SuppressantAminoglutethimide Breast Hormones and Acute and chronic lymphocyticAntagonists costeroids leukemias, non-Hodgkin's lymphomas, Hodgkin'sdisease, breast Progestins Hydroxy- Endometrium, breast progesteronecaproate Medroxy- progesterone acetate Megestrol acetate EstrogensDiethylstil- Breast, prostate bestrol Ethinyl estradiol AntiestrogenTamoxifen Androgens tosterone propionate Fluoxymesterone AntiandrogenFlutamide Prostate Gonadotropin- Leuprolide Prostate, Estrogen-receptor-Releasing Goserelin positive breast hormone analog # incorporated byreference herein, in particular for treatment protocols.

[0089] 3-aryloxy-3-phenylpropylamine compounds, methods for making sameand methods for using them are described in U.S. Pat. Nos. 4,018,895,4,314,081, 5,166,437 and 6,258,853 which are incorporated by referenceherein, and further below.

[0090] 3-Aryloxy-3-phenylpropylamines used as chemosensitizers may beadministered before, together with or after administration of thechemotherapeutic agent. Preferably, the 3-aryloxy-3-phenylpropylamineand the chemotherapeutic agent are administered substantially at thesame time.

[0091] The administration of a chemosensitizer and a chemotherapeuticagent substantially at the same time is a highly and advantageousfeature in the treatment of multidrug resistance (MDR) cells.

[0092] As is discussed in detail hereinabove, MDR is effected via anextrusion mechanism, which involves energy-dependant extrusion channelsor pumps that actively pump the drug out of the cells, thereby reducingits intracellular concentration below lethal threshold. As is furtherdiscussed hereinabove, chemosensitizers in this respect are agents thatreverse or modulate multidrug resistance in MDR cells by modulating theactivity of the MDR extrusion pumps. It is therefore advantageous thatthe chemosensitizing agent and the chemotherapeutic agent would beadministered substantially at the same time, in order to allow theircombined action by their dual presence in the treated cell.

[0093] Hence, the phrase “substantially at the same time”, as usedherein, means that the 3-aryloxy-3-phenylpropylamine and thechemotherapeutic agent are administered in such time intervals thatwould allow their dual presence in effective concentrations in thetreated cells. The 3-aryloxy-3-phenylpropylamine and thechemotherapeutic agent can be administered by different or identicalroutes of administration.

[0094] The 3-aryloxy-3-phenylpropylamine may be administered as a singledose, or it may be administered as two or more doses separated by a timeinterval. Where the 3-aryloxy-3-phenylpropylamine is administered as twoor more doses, the time interval between the3-aryloxy-3-phenylpropylamine administrations may be from about oneminute to about 12 hours, preferably from about 5 minutes to about 5hours, more preferably about 4 to 5 hours. The dosing protocol may berepeated; from one to three times, for example. Administration may beintravenous, intraperitoneal, parenteral, intramuscular, subcutaneous,oral, or topical, with oral and intravenous administration beingpreferred, and intravenous administration being presently mostpreferred.

[0095] The 3-aryloxy-3-phenylpropylamine used in the method of theinvention administered in a chemosensitizing effective amount.

[0096] As used herein the phrase “chemosensitizing effective amount”means that daily dose which results in an enhanced toxicity by achemotherapeutic agent, without adverse side effects. The specific dailydose will vary depending on the particular 3-aryloxy-3-phenylpropylamineused, the dosing regimen to be followed, and the particularchemotherapeutic agent with which it is administered. Such a daily dosecan be determined without undue experimentation by methods known in theart or as described herein.

[0097] As is exemplified in the Examples section that follows, apresently preferred chemosensitizing effective amount, according to thepresent invention, ranges between 0.05 mg/M² and 20 mg/M², preferablybetween 0.1 mg/M² and 10 mg/M², more preferably between 0.1 mg/M² and 7mg/M2, more preferably between 0.1 mg/M² and 5 mg/M2 and most preferablybetween 0.4 mg/M² and 4 mg/M².

[0098] The chemosensitizing effect of fluoxetine as a representativeexample of a 3-aryloxy-3-phenylpropylamine, was tested in vitro byadministering fluoxetine, in combination with different chemotherapeuticdrugs, at doses that range between 2 μM and 15 μM, administered everytwo days. These in vitro doses correspond to human daily doses thatrange between about 0.45 mg/M² and about 3.5 mg/M², respectively.

[0099] The conversion of these in vitro doses to in vivo human dailydoses is performed by first converting the μM fluoxetine concentrationsto units of mg/ml, and thereafter estimating the corresponding humandaily dose in mg/Kg body weight, taking into account that an averagehuman weight is about 70 Kg, an average human height is about 1.75 m andthat the human blood volume is 5 liters, and further taking into accountthat the in vitro treatment in the experiments conducted included asingle dose ever) two days. The obtained results are then converted intomg/M² surface area units, assuming that an individual weighting 70 Kgand of a height of 1.75 m has 1.85 M² skin surface.

[0100] In the in vivo studies, which are also described in detail in theExamples section that follows, the daily doses of fluoxetine were about0.04 mg/kg body, which correspond to 2.8 mg per 70 Kg body or to 1.5mg/M², according to the conversion index described hereinabove.

[0101] The chemosensitizing effective amount of fluoxetine as arepresentative 3-aryloxy-3-phenylpropylamine, as is demonstrated by thein vitro and in vivo studies described herein, is therefore well belowfluoxetine doses that are used in its classical, psychiatricindications, and are well below both its safety limit and its sideeffect-free limit. As is further shown in these in vitro and i17 vivostudies, at this low dose, fluoxetine has no anticancer effect and noadverse side effects.

[0102] The fact that fluoxetine exerts chemosensitizing effect in thetreatment of multidrug resistance cancer at such low doses is novel andhighly advantageous.

[0103] As is discussed in detail hereinabove, a method that involvesadministration of fluoxetine for treating cancer is described in WO94/19861.

[0104] The fluoxetine is used in this method as a compound that inhibitsproliferation of normal cells, while promoting the proliferation ofcancer cells, via histamine receptor antagonism mechanism. The fluxetinedose required to achieve these effects, according to the teachings of WO94/19861, is 20-40 mg/M², which is about 10-20 fold higher than the doseused in context of the present invention and about 2 fold higher thanthe side effect-free limit determined for fluxetine. As is furtherindicated in WO 94/18961, at this dose range, fluoxetine promotes theproliferation of cancer cells, and is further accompanied by adverseside effects such as cardiac arrhythmia. Also, administration offluoxetine according to the teachings of WO 94/18961 should precede theadministration of a chemotherapeutic agent in order to be effective. Insharp contrast, the administration of fluoxetine and thechemotherapeutic agent, according to the present invention, should besubstantially at the same time, for reasons set forth hereinabove.

[0105] Hence, the present invention provides a method of treating cancerthat is superior over to the presently known methods, as it involves theadministration of the chemosensitizing agent at low concentrations,within its safety and side effect-free limits, and therefore does notresults in adverse side effects.

[0106] A 3-aryloxy-3-phenylpropylamine for use as a chemosensitizeraccording to the teachings of the present invention may have structureI:

[0107] wherein each R′ is independently hydrogen or methyl

[0108] R is naphthyl or

[0109] R″ and R′″ are halo, trifluoromethyl, C₁-C₄ alkyl, C₁-C₃ alkoxyor C₃-C₄ alkenyl; and

[0110] n and m are 0, 1 or 2; and acid addition salts thereof formedwith pharmaceutically acceptable acids.

[0111] In the above formula when R is naphthyl, it can be eitheralpha-naphthyl or beta-naphthyl. R″ and R′″ when they are halo, C₁-C₄alkyl, C₁-C₃ alkyloxy or C₃-C₄ alkenyl represent, illustratively, thefollowing atoms or groups: fluoro, cliloro, bromo, iodo, methyl, ethyl,isopropyl, n-propyl, n-butyl. isobutyl, sec-butyl, t-butyl, methoxy,ethoxy, n-propoxy, isopropoxy, allyl, methallyl, crotyl and the like. Rthus can represent o, m and p-trifluoromethylphenyl, o, m andp-chlorophenyl, o, m and p-bromophenyl, o, m and p-fluorophenyl, o, mand p-tolyl, xylyl including all position isomers, o, m and p-anisyl, o,m and p-allylphenyl, o, m and p-methylallylphenyl, o, m andp-phenetolyl(ethoxyphenyl), 2,4-dichlorophenyl, 3,5-difluorophenyl,2-methoxy-4chlorophenyl, 2-methyl-4-chlorophenyl, 2-ethyl-4-bromophenyl,2,4,6-trimethylphenyl, 2-fluoro-4-trifluoromethylphenyl,2,4,6-trichlorophenyl, 2,4,5-trichlorophenyl and the like.

[0112] Also included within the scope of this invention are thepharmaceutically acceptable salts of the amine bases represented by theabove formula formed with non-toxic acids. These acid addition saltsinclude salts derived from inorganic acids such as: hydrochloric acid,nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydriodicacid, nitrous acid, phosphorous acid and the like, as well as salts ofnon-toxic organic acids including aliphatic mono and dicarboxylates,phenyl-substituted alkanoates, hydroxy alkanoates and alkanedioates,aromatic acids, aliphatic and aromatic sulfonic acids etc. Suchpharmaceutically-acceptable salts thus include: sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, nitrate, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, fluorodide, acetate,propionate, decanoate, caprylate, acrylate, formate, isobutyrate,caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, terephthalate,benzenesulfonates, toluenesulfonate, chlorobenzenesulfonate,xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate,citrate, lactate, beta-hydroxybutyrate, glycollate, malate, tartrate,methanesulfonate, propanesulfonates, naphthalene-1-sulfonate,naphthalene-2-sulfonate, mandelate and the like salts.

[0113] Compounds illustrative of the scope of this invention include thefollowing:

[0114] 3-(p-isopropoxyphenoxy)-3-phenylpropylamine methanesulfonate:

[0115] N,N-dimethyl 3-(3′,4′-dimethoxyphenoxy)-3-phenylpropylaminep-hydroxybenzoate;

[0116] N,N-dimethyl 3-(alpha-naphthoxy)-3-phenylpropylamine bromide;

[0117] N,N-dimethyl 3-(beta-naphthoxy)-3-phenyl-1-methylpropylamineiodide;

[0118] 3-(2′-methyl-4′,5′-dichlorophenoxy)-3-phenylpropylamine nitrate;

[0119] 3-(p-t-butylphenoxy)-3-phenylpropylamine glutarate;

[0120] N-methyl 3-(2 ′-chloro-p-tolyloxy)-3-phenyl-1-methylpropylaminelactate;

[0121] 3-(2′,4′-dichlorophenoxy)-3-phenyl-2-methylpropylamine citrate;

[0122] N,N-dimethyl 3-(m-anisyloxy)-3-phenyl-1-methylpropylaminemaleate;

[0123] N-methyl 3-(p-tolyloxy)-3-phenylpropylamine sulfate;

[0124] N,N-dimethyl 3-(2′,4′-difluorophenoxy)-3-phenylpropylamine2,4-dinitrobenzoate;

[0125] 3-(o-ethylphenoxy)-3-phenylpropylamine dihydrogen phosphate;

[0126]N-methyl-(2′-chloro-4′-isopropylphenoxy)-3-phenyl-2-methylpropylaminemaleate;

[0127] N,N-dimethyl 3-(2 ′-alkyl-4′-fluorophenoxy)-3-phenylpropylaminesuccinate;

[0128] N,N-dimethyl 3-(o-isopropoxyphenoxy)-3-phenyl-propylaminephenylacetate;

[0129] N,N-dimethyl 3-(o-)bromophenoxy)-3-phenyl-propylaminebeta-phenylpropionate;

[0130] N-methyl 3-(p-iodophenoxy)-3-phenyl-propylamine propiolate;

[0131] N-methyl 3-(3-n-propylphenoxy)-3-phenyl-propylamine decanoate;and preferably,

[0132] N-methyl 3-(p-trifluoromethylphenoxy)-3-phenylpropylamine.

[0133] The 3-aryloxy-3-phenylpropylamines of this invention in the formof their free bases are high boiling oils, but white crystalline solidsin the form of their acid addition salts. The compounds can be preparedin several ways. A particularly useful procedure for preparing compoundsrepresented by the above formula (in which both R′ groups attached tothe nitrogen are methyl) involves the reduction ofbeta-dimethylaminopropiophenone produced by a Mannich reaction to yieldN,N-dimethyl 3-phenyl-3-hydroxypropylamine. Replacement of the hydroxylgroup with a halogen, such as chlorine, yields the correspondingN,N-dimethyl 3-phenyl-3-chloropropylamine. Reaction of this chlorocompound with a suitably substituted phenol, as for exampleo-methoxyphenol (guiacol), produces a compound of this invention inwhich both R′ groups are methyl. Treatment of the N,N-dimethyl compoundwith cyanogenbromide serves to replace one N-methyl group with a cyanogroup. Hydrolysis of the resulting compound with base yields a compoundof this invention in which only one R′ group on the nitrogen is methyl.For example, treatment of N,N-dimethyl3-(o-anisyloxy)-3-phenylpropylamine with cyanogen bromide followed byalkaline hydrolysis of the N-cyano compound yields directly N-methyl3-(o-anisyloxy)-3-phenylpropylamine [N-methyl 3-(o-methoxyphenoxy)-3-phenylpropylamine].

[0134] An alternate preparation of the compounds of this invention inwhich only one of the R′ groups attached to the nitrogen is methyl iscarried as follows:

[0135] 3-Chloropropylbenzene is reacted with a positive halogenatingagent such N-bromosuccinimide to yield the corresponding3-chloro-1-bromopropylbenzene. Selective replacement of the bromo atomwith the sodium salt of a phenol, as for example, the sodium salt ofo-methoxyphenol (guiacol) yields a3-chloro-1-(]-methoxyphenoxy)-propylbenzene [also named as3-chloro-1-(o-anisyloxy)propylbenzene]. Reaction of the 3-chloroderivative thus produced with methylamine yields the desired N-methyl3-(o-anisyloxy)-3-phenylpropylamine.

[0136] 3-Aryloxy-3-phenylpropylamine compounds in which both R′ groupsattached to the nitrogen in the above formula are hydrogen can beprepared from an intermediate produced in the previous preparation ofthe N-methyl compounds such as, for illustrative purposes,3-chloro-1-(o-anisyloxy)-propylbenzene prepared by the reaction of3-chloro-1-bromobenzene and sodium guiacol. This chloro compound isreacted with sodium azide to give the corresponding3-azido-1-(o-anisyloxy)-propylbenzene. Reduction of the azide group witha metallo-organic reducing agent such as sodium borohydride yields thedesired primary amine. Alternatively, the chloro compound can be reacteddirectly with a large excess of ammonia in a high pressure reactor togive the primary amine.

[0137] 3-Aryloxy-3-phenylpropylamine compounds in which the R′ group onthe carbon atom alpha to the nitrogen is methyl can be prepared byreacting phenyl 2′-propenyl ketone with dimethylamine [See J. Am. Chem.Soc., 75, 4460 (1953)]. The resulting 3-dimethylaminobutyrophenone isreduced to yield the N,N-dimethyl3-hydroxy-1-methyl-3-phenylpropylamine. Replacement of the hydroxyl withchlorine followed by reaction of the chloro-compound with the sodiumsalt of a suitably substituted phenol yields the N,N-dimethylderivatives of this invention bearing an alpha methyl group on thepropylamine backbone of the molecule. Production of the correspondingN-methyl derivative can be accomplished by the aforementioned reactionsequence utilizing cyanogen bromide. The N-methyl derivative can in turnbe converted to the corresponding primary amine (in which both R′ groupson the nitrogen are hydrogen) by oxidation in neutral permanganateaccording to the procedure of Booher and Pohland, Ser. No. 317,969,filed Dec. 26, 1972. Compounds in which the R′ group attached to thebeta-carbon atom is methyl are prepared by a Mannich reaction involvingpropiophenone, formaldehyde and dimethylamine. The resulting ketone, analpha-methyl-beta-dimethylaminopropiophenone, is subjected to the samereduction procedure as before to yield a hydroxy compound. Replacementof the hydroxyl with chlorine followed by reaction of the chlorocompound with the sodium salt of a phenol yields a dimethyl aminecompound of this invention. Conversion of the dimethylamine to thecorresponding monomethyl and primary amines is carried out as before.

[0138] Those 3-aryloxy-3-phenylpropylamine compounds in which the R′group attached to either the alpha or beta-carbon is methyl have twoasymmetric carbon atoms, the carbon carrying the R′ methyl and the.gamma.-carbon carrying the phenoxy and phenyl groups. Thus, suchcompounds exist in four diastereomeric forms occurring as two racemicpairs, the less soluble pair being designated alpha-dl form and the moresoluble the beta-dl form. Each racemate can be resolved into itsindividual d and l isomers by methods well known in the art,particularly, by forming salts with optically active acids andseparating the salts by crystallization.

[0139] A 3-aryloxy-3-phenylpropylamine may be coupled to asite-directing molecule to form a conjugate for targeted in vivodelivery. “Site-directing” means having specificity for targeted sites.“Specificity for targeted sites” means that upon contacting the3-aryloxy-3-phenylpropylamine -site-directing-conjugate with thetargeted site, for example, under physiological conditions of ionicstrength, temperature, pH and the like, specific binding will occur. Theinteraction may occur due to specific electrostatic, hydrophobic,entropic or other interaction of certain residues of the conjugate withspecific residues of the target to form a stable complex underconditions effective to promote the interaction. Exemplarysite-directing molecules contemplated in the present invention includebut are not limited to: oligonucleotides, polyamides including peptideshaving affinity for a biological receptor and proteins such asantibodies; steroids and steroid derivatives; hormones such asestradiol, or histamine; hormone mimics such as morphine; and furthermacrocycles such as sapphyrins and rubyrins.

[0140] As used herein, a “site-directing molecule” may be anoligonucleotide, an antibody, a hormone, a peptide having affinity for abiological receptor, a sapphyrin molecule, and the like. A preferredsite-directing molecule is a hormone, such as estradiol, estrogen,progesterone, and the like. A site-directing molecule may have bindingspecificity for localization to a treatment site and a biologicalreceptor may be localized to a treatment site. A3-aryloxy-3-phenylpropylamine oligonucleotide-conjugate, where theoligonucleotide is complementary to an oncogenic messenger RNA, forexample, would further localize chemotherapeutic activity to aparticularly desired site. Antisense technology is discussed in U.S.Pat. Nos. 5,194,428, 5,110,802 and 5,216,141, all of which areincorporated by reference herein.

[0141] A couple may be described as a linker, i.e., the covalent productformed by reaction of a reactive group designed to attach covalentlyanother molecule at a distance from the 3-aryloxy-3-phenylpropylaminemacrocycle. Exemplary linkers or couples are amides, amine, thiol,thioether, ether, or phosphate covalent bonds. In most preferredembodiments, site-directing molecules are covalently bonded to the3-aryloxy-3-phenylpropylamine via a carbon-nitrogen, carbon-sulfur, or acarbon-oxygen bond.

[0142] Generally, water soluble 3-aryloxy-3-phenylpropylamines retaininglipophilicity are preferred for the applications described herein.“Water soluble” means soluble in aqueous fluids to about 1 mM or better.“Retaining lipophilicity” means having greater affinity for lipid richtissues or materials than surrounding nonlipid rich tissues. “Lipidrich” means having a greater amount of triglyceride, cholesterol, fattyacids or the like.

[0143] Representative examples of useful steroids include any of thesteroid hormones of the following five categories: progestins (e.g.progesterone), glucocorticoids (e.g., cortisol), mineralocorticoids(e.g., aldosterone), androgens (e.g., testosterone) and estrogens (e.g.,estradiol).

[0144] Representative examples of useful amino acids of peptides orpolypeptides include amino acids with simple aliphatic side chains(e.g., glycine, alanine, valine, leucine, and isoleucine), amino acidswith aromatic side chains (e.g., phenylalanine, tryptophan, tyrosine,and histidine), amino acids with oxygen and sulfur-containing sidechains (e.g., serine, threonine, methionine, and cysteine), amino acidswith side chains containing carboxylic acid or amide groups (e.g.,aspartic acid, glutamic acid, asparagine, and glutamine), and aminoacids with side chains containing strongly basic groups (e.g., lysineand arginine), and proline. Representative examples of useful peptidesinclude any of both naturally occurring and synthetic di-, tri-, tetra-,pentapeptides or longer peptides derived from any of the above describedamino acids (e.g., endorphin, enkephalin, epidermal growth factor,poly-L-lysine, or a hormone). Representative examples of usefulpolypeptides include both naturally occurring and synthetic polypeptides(e.g., insulin, ribonuclease, and endorphins) derived from the abovedescribed amino acids and peptides.

[0145] The term “a peptide having affinity for a biological receptor”means that upon contacting the peptide with the biological receptor, forexample, under appropriate conditions of ionic strength, temperature, pHand the like, specific binding will occur. The interaction may occur dueto specific electrostatic, hydrophobic, entropic or other interaction ofcertain amino acid or glycolytic residues of the peptide with specificamino acid or glycolytic residues of the receptor to form a stablecomplex under the conditions effective to promote the interaction. Theinteraction may alter the three-dimensional conformation and thefunction or activity of either or both the peptide and the receptorinvolved in the interaction. A peptide having affinity for a biologicalreceptor may include an endorphin, an enkephalin, a growth factor, e.g.epidermal growth factor, poly-L-lysine, a hormone, a peptide region of aprotein and the like. A hormone may be estradiol, for example.

[0146] For use as a chemosensitizer, 3-aryloxy-3-phenylpropylamines areprovided as pharmaceutical preparations. A pharmaceutical preparation ofa 3-aryloxy-3-phenylpropylamine may be administered alone or incombination with pharmaceutically acceptable carriers, in either singleor multiple doses. Suitable pharmaceutical carriers include inert soliddiluents or fillers, sterile aqueous solution and various organicsolvents. The pharmaceutical compositions formed by combining a3-aryloxy-3-phenylpropylamine of the present invention and thepharmaceutically acceptable carriers are then easily administered in avariety of dosage forms such as injectable solutions.

[0147] For parenteral administration, solutions of the3-aryloxy-3-phenylpropylamine in sesame or peanut oil, aqueous propyleneglycol, or in sterile aqueous solution may be employed. Such aqueoussolutions should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure.

[0148] The pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy use with a syringe exists. It must be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms, such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars such as mannitol or dextrose or sodiumchloride. A more preferable isotonic agent is a mannitol solution ofabout 2-8% concentration, and, most preferably, of about 5%concentration. Prolonged absorption of the injectable compositions canbe brought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

[0149] Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0150] As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutically active substancesis well known in the art. Except insofar as any conventional media oragent is incompatible with the active ingredient, its use in thetherapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

[0151] 3-Aryloxy-3-phenylpropylamines may be co-formulated with achemotherapeutic agent. Methods of co-formulating more than a singleactive ingredient are well known in the art. Such co-formulation ensuresco-administration of the chemotherapeutic agent and the3-aryloxy-3-phenylpropylamine.

[0152] Hence pharmaceutical compositions that comprise a3-aryloxy-3-phenylpropylamine, as a chemosensitizing agent, and achemotherapeutic agent, are provided in accordance with the presentinvention. Such pharmaceutical compositions can be formulated asdescribed hereinabove.

[0153] The pharmaceutical compositions of the present invention may bepresented in a pack or dispenser device, such as a FDA approved kit,which may contain one or more unit dosage forms containing the activeingredients. The package may, for example, comprise metal or plasticfoil, such as a blister package. The package or dispenser device may beaccompanied by instructions for administration and indication. Thepackage or dispenser may also be accompanied by a notice associated withthe container in a form prescribed by a governmental agency regulatingthe manufacture, use or sale of pharmaceuticals, which notice isreflective of approval by the agency of the form of the compositions orhuman or veterinary administration. Such notice, for example, may be oflabeling approved by the U.S. Food and Drug Administration forprescription drugs or of an approved product insert.

[0154] The pharmaceutical compositions of the present invention cantherefore be packaged in a packaging material and identified in print inor on the packaging material for use in the treatment of a multi drugresistance cancer.

[0155] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

[0156] Hence, additional objects, advantages, and novel features of thepresent invention will become apparent to one ordinarily skilled in theart upon examination of the following examples, which are not intendedto be limiting. Additionally, each of the various embodiments andaspects of the present invention as delineated hereinabove and asclaimed in the claims section below finds experimental support in thefollowing examples.

EXAMPLES

[0157] Reference is now made to the following examples, which togetherwith the above descriptions, illustrate the invention in a non limitingfashion.

In vitro Studies Materials and Methods

[0158] Chemotherapies (CT):

[0159] Mitomycin C (MMC), Vinblastine (VIN) and Doxorubicin (DOX).

[0160] Chemosensitizer (CS):

[0161] Fluoxetine.

[0162] Cell Lines:

[0163] MCF-7 (human breast carcinoma), HT1080 (human fibrosarcoma), U2OS(human osteosarcoma), PANC-1 (human pancreatic adenocarcinoma), C6 (ratglioblastoma), C26 (murine colon adenocarcinoma), and B16F10 (murinemelanoma).

[0164] Cell Culture Growth and Maintenance Media:

[0165] Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%fetal calf serum (FCS), Penicillin (10,000 units/ml), Streptomycin (10mg/ml) and L-Glutamine (200 mM).

[0166] Cell Cultures:

[0167] Cells were grown in monolayers in 100×20 mm dishes, in the growthmedia listed above, at 37° C. in 5% CO₂.

[0168] Cell Survival:

[0169] Cells were grown in monolayers as describe above and seeded onto96 multiwell plates at a density of 1×10⁴ cells/ml, 24 hours prior to anexperiment. Twenty four hours later, the media was replaced by treatmentmedia as is detailed in the Experimental Results section that follows.The experiments were terminated 24 or 48 hours post media replacement.The quantity of viable cells was determined by the MTT test, recordingthe absorbencies in a plate reader, at two wavelengths: 550 and 650 nm.

[0170] Drug Efflux Measurements:

[0171] Cells were grown in monolayers as described above. Several daysprior to an experiment the cells (at a density in the range of5×10⁴-5×10 ⁵ cells/ml) were seeded into 24 multiwell culture plates. Theexperiments were performed when the cells reached semi-confluency. Theefflux experiments were conducted according to the following protocol:Cells were loaded, by incubation for 10 hours, with either a nonlethaldose of DOX, or the same DOX dose combined with fluoxetine. Controlcells received media alone. At the end of the incubation, the media wasremoved, the cells washed with buffer, and thereafter incubated witheither media alone (for the wells incubated with DOX only) or media withfluoxetine at the same dose as in the 10 hours incubation. At selectedtime points, the medium from every well was collected and replaced witha fresh similar medium. At the end of the experiment the cells in eachwell were dissolved by adding 5% Deoxycholate (DOC) to each well.Samples from the media collected at each time point, and samples fromthe final detergent-dissolved cells, were transferred to a 96 well platesuitable for a fluorimeter plate reader. Excitation and emission were at480 nm and 530 nm, respectively. Calibration curves were run with eachassay using DOX standards dissolved in the appropriate media (i.e.,buffer or buffer/DOC).

[0172] Drug Efflux Measurements Using Rhodamine-123:

[0173] Rhodamine-123 is a well-known substrate of Pgp, the first MDRextrusion channel identified, and hence serves as a fluorescentindicator for the presence and activity of a chemosensitizer. As theaccumulation of Rhodamine-123 in Pgp-containing multidrug resistancecells is increased significantly in the presence of a chemosensitizer,the effects of a molecule suspected to inhibit MDR pumps on theintracellular accumulation of Rhodamine-123 has become a classical testfor chemosensitizers.

[0174] The effect of fluoxetine as a chemosensitizer was thereforemeasured and compared to the known chemosensitizer Verapamil, byRhodamine-123 fluorescence measurements. The accumulation ofRhodamine-123 was measured on C-26 cells, which are known asPgp-containing cells, in two different systems: In system 1, suspensionsof C-26 cells were used and the accumulation was measured by flowcytometry; In system II, adherent monolayers of C-26 cells were used andthe accumulation was measured by confocal microscopy., according to thefollowing protocols:

[0175] System I: Suspensions of C-26 cells in PBS (1×10⁶ cells/ml) wereincubated for 30 minutes, at 37° C., with 5 μM Rhodamine-123 alone andin combination with fluoxetine, at varying doses. For comparison,experiments were performed also with Verapamil as a chemosensitizer, ata typical dose of 15 μM. The cells were thereafter analyzed forintracellular fluorescence, using a flow cytometer (Becton-Dickinson,USA). Excitation and emission were at 485 nm and 547 nm, respectively.

[0176] System II: C-26 cells were grown as monolayers on cover slides,and were thereafter incubated for one hour at 37° C., with 5 μMRbodamine-123 alone, an in combination with a chemosensitizer asdescribed hereinabove. At the end of the incubation time, the cells werewashed 6 times with PBS, fixed with a mounting medium (Mounting mediumwith anti-fading agents, Biomeda corp., CA, USA) and were analysed byConfocal microscopy (Zeiss LSM 510). Excitation was at 488 nm andemission was measured using a band pass filter of 505-550 nm.

Experimental Results

[0177] Testing the Response of MDR Cell Lines to a Single FluoxetineDose:

[0178] C6 and, in separate experiments, PANC-1 cells were seeded ontomultiwell (96) culture plates, and the experiments were initiated whenthe cells reached semi-confluency. The serum-supplemented cell growthmedia was replaced by a treatment media, selected from: (i) thecombination of a chemotherapeutic drug and fluoxetine dissolved in serumsupplemented growth media; (ii) chemotherapeutic drug dissolved in serumsupplemented growth media (iii) fluoxetine dissolved in serumsupplemented growth media; and (iv) serum supplemented growth mediaalone (untreated control). Drug species and dose were the same in (i)and (ii). Fluoxetine dose was the same in (i) and (iii). The experimentwas terminated 24 hours later, and the number of viable cells wasquantitated using the MTT method.

[0179] Typical results, showing the effects of the various treatmentgroups on cell death are shown in FIGS. 1 and 2, for both cell lines andfor the chemotherapeutic drugs Mitomycin C (MMC) and Doxorubicin (DOX),respectively. The fluoxetine dose used in these experiments matches thehighest safe dose used for accepted indications of fluoxetine. The dataclearly shows that fluoxetine alone does not affect cell survival atall. At the drug doses applied (listed in FIGS. 1 and 2), treatment withdrug alone was only mildly effective in causing cell death, at its bestno more than 20%. In contrast, for the four cases studied (2 drugs, 2cell lines), the combination treatment caused a significant enhancementin cell death, which was 3-4 fold, clearly showing the effectiveness ofthe combination treatment.

[0180] These experiments were similarly performed with B16F10 and C-26cell lines, which are known as drug resistance cells. Mitomycin C andDoxorubicin were applied, at typical doses of 50 μg/ml and 1.0 μg/ml,respectively, with and without 15 μM of fluoxetine. The treatment withthe drug alone generated cell death in the range of 10-15%, thusconfirming the inherent-resistant nature of these cell lines. Treatmentwith a combination of the drug and fluoxetine increased the cell deathto about of 80-90%, thus demonstrating the increased cell demise causedby the chemosensitizer.

[0181] Evaluating Fluoxetine Dose Response:

[0182] Studies similar in general to those outlined in the previoussection, were conducted with five cell lines selected for this task(PANC-1, C6, MCF-7, U2OS and HT1080), increasing the length of theexperiment to 48 hours. The studies were done with DOX and withVinblastine (VIN or VLB). The treatment groups were similar to thoselisted in the previous sections, with the following additions: a seriesof fluoxetine doses were tested, alone and in combination with thecytotoxic drugs, covering a fluoxetine range of 0-15 μM.

[0183] As expected from the testing with 15 μM, fluoxetine alone was nottoxic to the cells. For the drug species and respective doses tested—0.1μg/ml DOX and 0.3 μg/ml VIN—drugs alone caused 50% and 10-20% reductionsin cell survival, for the non-resistant and resistant cell lines,respectively. Normalizing, for each cell line, the survival of cellsreceiving the chemotherapeutic drug and fluoxetine, to the survival ofthe cells receiving the chemotherapeutic drug alone (i.e., zerofluoxetine) it was possible to construct fluoxetine dose responsecurves. A typical example is shown in FIG. 3, for the PANC-1 cell line,with both drugs. From such dose response curves, using computer-aidedpolynomial curve fitting, it was possible to determine for each drug andeach cell line, an LD₅₀ for the fluoxetine potentiation effect. TheseLD₅₀ values, for all five cell lines, each with both drugs, are shown inFIG. 4.

[0184] Several features of these results are worthy of attention:

[0185] First, in all cases fluoxetine potentiates the cytotoxic effectof the chemotherapeutic drug.

[0186] Second, the LD₅₀ range, which spans from 7-10 μM fluoxetine and6.5-8 μM fluoxetine, for DOX and VIN, respectively, is well below thehighest safety limit of 15 μM fluoxetine. This is completely differentthan the cases of Verapamil and Cyclosporin, where the dose range forchemosensitization was well above their safety limit and henceimpractical for clinical applications.

[0187] Third, taking into consideration that in the resistant cell linesthe potentiation has to work on double the number of cells than in thenon-resistant lines, yet the LD₅₀ range is quite similar—these dataimply that the potentiation effect is more significant in the MDR lines.

[0188] Fourth, in the non-resistant lines, the effect of fluoxetine on agiven line is not drug-sensitive while in the resistant lines,fluoxetine is more potent (lower LD₅₀) with VIN than with DOX.

[0189] Insights and Results with Respect to the Operating Mechanisms:

[0190] Without an intention to limit the present invention in any way,the data presented herein allows speculating some mechanistic insightswith respect to the chemosensitization activity of3-aryloxy-3-phenylpropylamine in general and fluoxetine in particular.

[0191] The finding that fluoxetine potentiates the cytotoxicity withdifferent drugs, that have furthermore different killing mechanisms,rules out a drug-specific effect. Could fluoxetine be triggering a celldeath mechanism that it totally independent of the presence of thechemotherapeutic drug in the cell? It is suggest this triggering optionis unlikely in view of the finding that fluoxetine alone is not toxic tothe cells—at the same dose level where it exerts its effect in thepresence of a cytotoxic drug. Since the sites of action for thechemotherapeutic drugs are intracellular it is reasonable to assume thatfluoxetine exerts its effect(s) inside the cell, also.

[0192] In general, nature has not planned for the introduction offoreign matter such as drugs, into living biological systems. Hencenature has made no specific efforts to assist drug entry into cells.That drugs do gain entry into cells is a fact of life. Drugs do it by atleast two pathways that are not mutually exclusive: (i) by diffusionacross the cell membrane, driven by the drug's electrochemical-potentialgradient; and (ii) by “borrowing a ride” on natural transport systemsdesigned (by nature) to transport molecules that are a normal componentof a living system. Obviously both pathways can operate in bothdirections, namely influx and efflux. In addition, the interaction ofthe foreign entity with biological transport systems can take the formof blockage, where a foreign matter blocks the passage of othermaterials through the transporter.

[0193] The data presented herein reveal that fluoxetine acts on both MDRand non-multidrug resistance cells, but is more effective with theformer type. This raises at least two possibilities for fluoxetine'smechanism(s) of action:

[0194] First: Fluoxetine inhibits extrusion channels that pumpchemotherapeutic drugs out of the cells, reducing the intracellular drugdoses below the lethal threshold. The fact that both MDR andnon-multidrug resistance cells have been affected, but to differentextent, fits with the extrusion pumps being natural proteins that canexist in all cells, but in significantly larger numbers (copies percell) in multidrug resistance cells.

[0195] Second: Fluoxetine has two different activities: One is pumpinhibition as above especially (and possibly only) in the multidrugresistance cells. The other is enhancement of the cellular response tothe chemotherapeutic drug, without any change in the intracellular druglevel. The latter could operate in the non-multidrug resistance cellsalone, or in both types of cells.

[0196] An experimental method to support or refute the first mechanism,is the following:

[0197] Cells are loaded with non-lethal doses of a chemotherapeutic drugalone, or drug and fluoxetine. Upon completion of loading theextracellular fluid is replaced with buffer alone, and the efflux ofdrug into the external media is monitored for several hours. Iffluoxetine inhibits efflux pumps, drug efflux in the systems receivingthe combined treatment should be slower than in those receiving the drugalone. This expectation was met, as shown by the following:

[0198] The effect of fluoxetine on DOX efflux from C6 cells was studiedas detailed under the Methods section above. The DOX and fluoxetineloading doses were 0.1 μg/ml and 15 μM, respectively. The cumulativequantity of DOX that diffused out of the cells at time=t was normalizedto the total intracellular concentration of DOX at time=0, and isdenoted f(t). The magnitudes of f(t) as function of time are plotted inFIG. 6, for the cells that received DOX alone and for the cells thatreceived DOX with fluoxetine.

[0199] The data presented makes it clear that 2 hours suffice forcomplete depletion of intracellular DOX from cells that were loaded withDOX alone. In contrast, DOX efflux was significantly slower in cellsthat received both DOX and fluoxetine. At 2 hours loss of intracellularDOX (in the combined treatment) was under 40%, and complete depletionwas 450% slower than in the absence of fluoxetine. The pattern of DOXefflux from the cells loaded with this drug alone fits dominance of asingle pathway. Based on previous experience, were the efflux seen forthe DOX-alone systems dominated by self diffusion of the drug throughthe lipid bilayer membranes, at 2 hours f(t) would range from 10-30%.This clearly indicates that the single efflux pathway, that provides100% depletion at 2 hours, can be assigned to an extrusion pump.

[0200] DOX efflux from cells that received the combined treatment is atthe least bi-phasic, which indicates that DOX diffuses out of thosecells by at least two pathways. The pattern of the fastest pathway,which dominates efflux at the first 30 minutes and accounts for ≦20% ofthe total depletion, is quite similar to that of the DOX-alone case. Thepattern of the remaining 80% fits one or more additional, significantlyslower, pathways. These data imply a fluoxetine effect at the transportlevel, a major part of which is reduction in the number of active pumps,with possible minor effects of reduction in the rate constant of DOXefflux through this pump.

[0201] Support for these data was found in the drug efflux measurementswith Rhodamine-123, a fluorescent compound that serves as an indicatorfor the effect of a molecule on MDR extrusion pumps. As is detailedhereinabove in the methods section, the chemosensitizing effects offluxetine and Verapamil on the intracellular level of Rhodamine-123 weremeasured in two different systems.

[0202] As is shown in FIG. 7, in the experiments performed in suspendedcells, Verapamil, at the standard dose (15 μM), generated a minorincrease of 23% of the intracellular fluorescence, as compared to thecontrol, chemosensitizing-free cells, while fluoxetine, at the same dosegenerated an increase of 140% of the intracellular fluorescence. Theexperiments further showed a direct correlation between the fluoxetinedose and the fluorescence level. By comparing the results obtained withthe known chemosensitizer Verapamil and with fluoxetine, it is clearlydemonstrated that (i) fluoxetine acts as a chemosensitizer by exertingthe same effect as Verapamil on the intracellular fluorescence level;and (ii) the chemosensitizing potential of fluoxetine in substantiallyhigher than that of Verapamil.

[0203] Similar results were obtained in the experiments performed withmonolayered cells. As is shown in FIGS. 8a-c, while Verapamil generatedan increase in the intracellular level of Rhodamine-123 (FIG. 8b), ascompared to the control, chemosensitizer-free, cells (FIG. 8a),fluoxetine generated a substantially higher increase in theintracellular accumulation of Rhodamine-123 (FIG. 8c).

[0204] Hence, the results obtained by the Rhodamine-123 measurementsprovide additional support for the inhibitory effect of fluoxetine onMDR extrusion pumps, which was suggested upon the efflux studiesdescribed hereinabove. The chemosensitizing activity of fluoxetine inboth suspended and monolayered cells, provides an indication for its invivo chemosensitizing activity in both solid and non-solid tumors, as isfurther demonstrated hereinbelow.

In vivo Studies Materials and Methods

[0205] The following in vivo studies were conducted in mice, in twotumor models: a solid tumor model (also referred to herein as model 1)and a lung metastasis model (also referred to herein as model 2).

[0206] Cells:

[0207] In the solid tumor model, C-26 cells were injected into theanimal's right-hind footpad.

[0208] In the lung metastasis model, B16F10 cells were injectedintravenously, into the tail vein.

[0209] Chemosensitizer (CS):

[0210] The chemosensitizer used in both models was fluoxetine.

[0211] In both models the chemosensitizer was administered orally, viathe drinking water. Daily intake was 0.04 mg/kg body weight. This dailydose is equivalent to a daily dose of 2.8 mg for a human weighting 70kg, whereas the approved (safe) range of daily dose of fluoxetine as anantidepressant is 20-80 mg for a human weighting 70 kg.

[0212] Chemotherapeutic Agents (CT):

[0213] In the solid tumor model, Mitomycin C (MMC) and Doxorubicin(DOX), were tested separately, each at a dose of 5 mg/kg body.

[0214] In the lung metastasis model, only Doxorubicin, at a dose of 10mg/kg body, was tested.

[0215] In each model, the animals were divided into 4 groups, 5 animalsper group. In the lung metastatic model, a fifth group of untreated,healthy animals (i.e., animals that were not inoculated with tumorcells), served as a control group for both models.

[0216] Each of the animal groups was treated with saline,chemosensitizer, chemotherapeutic agent or a combination ofchemotherapeutic agent and chemosensitizer. The saline and thechemotherapeutic agent were injected into the tail vein (100 μl). Thedosing regimen in the solid tumor model experiments was 3 injections,spaced a week apart and starting at day 5 from tumor inoculation. Thedosing regimen in the lung metastatic model was also 3 injections, atdays 1, 5 and 9 from tumor inoculation.

Experimental Results

[0217] The Solid Tumor Model:

[0218] The effective impact of the combined treatment of fluoxetine anda chemotherapeutic agent in the solid tumor model is demonstrated inFIGS. 9 and 10. FIG. 9 clearly demonstrates that the solid tumorincreases fast and exponentially in the groups treated with saline, achemosensitizer alone and a chemotherapeutic agent alone. These resultsindicate that the tumor retains its drug resistance nature in vivo.Contrary to that, in the animals receiving the combination therapy, theappearance of the tumor is delayed, as compared with the other groups,the tumors are substantially smaller and the tumor growth issignificantly slower. FIG. 10 indicates the same trend, as itdemonstrates that only the animals receiving the combination therapywere long survivors, namely, the survival of the animals treated withMMC+CS and DOX+CS was prolonged 2 and 3 fold, respectively, as comparedwith animals treated with saline, CS alone and the respective CT alone.

[0219] The Lung Metastasis Model:

[0220] The results presented in FIGS. 11a and 11 b demonstrate the lungmetastatic burden by two measures: the increase in the lung weight (FIG.11a) and the number of lung metastasis (FIG. 11b). These results clearlyindicate that, by both measures, animals treated with saline or achemosensitizer alone had the highest metastatic burden. These resultsfurther demonstrate that treatment with Doxorubicin generated only amild reduction in the metastatic burden, while the combination treatmentof chemosensitizer and a Doxorubicin generated a substantial reductionthereof.

[0221] This encouraging effect of the combination treatment is reflectedalso in the survival results presented in FIG. 12. The survival data inFIG. 12 show the results obtained in a 75-days experiment. As is shownin FIG. 12, similar to the pattern of the solid tumor model (FIG. 10),animals treated with saline, CS alone or CT alone, died rather early andwithin short intervals of one another, while those treated with thecombination treatment were long survivors.

[0222] Of the two tumor models tested, the B16F10 is a more aggressivetumor. This fact is evident by the shift in the survival data in FIG.12, as compared with the data shown in FIG. 10, towards a shorter timespan between the administration of the tumor cells to the animals andonset of animal demise.

[0223] The obtained results clearly demonstrate the advantageousfeatures of fluoxetine, as a representative example of a3-aryloxy-3-phenylpropylamine, as a chemosensitizing agent, as isdelineated hereinbelow:

[0224] This chemosensitizing agent changes the course of the tumorresponse to chemotherapeutic drugs from poor to excellent, by allcounts: tumor progression, metastatic burden, and survival.

[0225] As the chemosensitizing activity of the CS agent was demonstratedwith two different chemotherapeutic drugs, acting via different pathways(as is discussed in detail hereinabove, this CS agent is not drugspecific and therefore has the potential to resolve the drug resistanceto additional drugs.

[0226] The chemosensitizer itself at the doses employed has nodetrimental effects with respect to tumor progression.

[0227] The dose range required for chemosensitization is well below thesafe dose in humans.

[0228] Finally, the chemosensitizer is administerable orally, which is apatient-friendly route of administration.

[0229] It is appreciated that certain features of the invention, whichare, for clarity, described in the context of separate embodiments, mayalso be provided in combination in a single embodiment. Conversely,various features of the invention, which are, for brevity, described inthe context of a single embodiment, may also be provided separately orin any suitable subcombination.

[0230] Although the invention has been described in conjunction withspecific embodiments thereof, it is evident that many alternatives,modifications and variations will be apparent to those skilled in theart. Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

What is claimed is:
 1. A method of treating a subject suspected ashaving, or having a multidrug resistance cancer, the method comprisingadministering to the subject a chemotherapeutically effective amount ofat least one chemotherapeutic agent and a chemosensitizing effectiveamount of at least one 3-aryloxy-3-phenylpropylamine.
 2. The method ofclaim 1, wherein said administering said at least one chemotherapeuticagent and said at least one 3-aryloxy-3-phenylpropylamine is performedsubstantially at the same time.
 3. The method of claim 1, wherein saidchemosensitizing effective amount ranges between about 0.1 mg/M² andabout 10 mg/M².
 4. The method of claim 1, wherein said cancer isselected from the group consisting of leukemia, lymphoma, carcinoma andsarcoma.
 5. The method of claim 1, wherein said at least one3-aryloxy-3-phenylpropylamine is administered orally.
 6. The method ofclaim 1, wherein said at least one 3-aryloxy-3-phenylpropylamine is ofthe formula:

wherein each R′ is independently hydrogen or methyl; R is naphthyl or

R″ and R′″ are halo, trifluoromethyl, C₁-C₄ alkyl, C₁-C₃ alkoxy or C₃-C₄alkenyl; and n and m are 0, 1 or 2; and acid addition salts thereofformed with pharmaceutically acceptable acids.
 7. The method of claim 1,wherein said at least one 3-aryloxy-3-phenylpropylamine is selected fromthe group consisting of 3-(p-isopropoxyphenoxy)-3-phenylpropylaminemethanesulfonate, N,N-dimethyl3-(3′,4′-dimethoxyphenoxy)-3-phenylpropylamine p-hydroxybenzoate,N,N-dimethyl 3-(alpha-naphthoxy)-3-phenylpropylamine bromide,N,N-dimethyl 3-(beta-naphthoxy)-3-phenyl-1-methylpropylamine iodide,3-(2′-methyl-4′,5′-dichlorophenoxy)-3-phenylpropylamine nitrate,3-(p-t-butylphenoxy)-3-phenylpropylamine glutarate, N-methyl3-(2′-chloro-p-tolyloxy)-3-phenyl-1-methylpropylamine lactate,3-(2′,4′-dichlorophenoxy)-3-phenyl-2-methylpropylamine citrate,N,N-dimethyl 3-(m-anisyloxy)-3-phenyl-1-methylpropylamine maleate,N-methyl 3-(p-tolyloxy)-3-phenylpropylamine sulfate, N,N-dimethyl3-(2′,4′-difluorophenoxy)-3-phenylpropylamine 2,4-dinitrobenzoate,3-(o-ethylphenoxy)-3-phenylpropylamine dihydrogen phosphate,N-methyl-(2′-chloro-4′-isopropylphenoxy)-3-phenyl-2-methylpropylaminemaleate, N,N-dimethyl 3-(2′-alkyl-4′-fluorophenoxy)-3-phenylpropylaminesuccinate, N,N-dimethyl 3-(o-isopropoxyphenoxy)-3-phenyl-propylaminephenylacetate, N,N-dimethyl 3-(o-)bromophenoxy)-3-phenyl-propylaminebeta-phenylpropionate, N-methyl 3-(p-iodophenoxy)-3-phenyl-propylaminepropiolate, N-methyl 3-(3-n-propylphenoxy)-3-phenyl-propylaminedecanoate, and N-methyl3-(p-trifluoromethylphenoxy)-3-phenylpropylamine.
 8. The method of claim1, wherein said at least one 3-aryloxy-3-phenylpropylamine is N-methyl3-(p-trifluoromethylphenoxy) -3-phenylpropylamine or a pharmaceuticallyacceptable salt thereof.
 9. The method of claim 1, wherein said at leastone chemotherapeutic agent is selected from the group consisting of analkylating agent, an antimetabolite, a natural product, a miscellaneousagent, a hormone and an antagonist.
 10. A method of treating a subjectsuspected as having, or having a multidrug resistance cancer, the methodcomprising administering to the subject, substantially at the same time,a chemotherapeutically effective amount of at least one chemotherapeuticagent and a chemosensitizing effective amount of at least one3-aryloxy-3-phenylpropylamine.
 11. The method of claim 10, wherein saidchemosensitizing effective amount ranges between about 0.1 mg/M² andabout 10 mg/M².
 12. The method of claim 10, wherein said cancer isselected from the group consisting of leukemia, lymphoma, carcinoma andsarcoma.
 13. The method of claim 10, wherein said at least one3-aryloxy-3-phenylpropylamine is administered orally.
 14. The method ofclaim 10, wherein said at least one 3-aryloxy-3-phenylpropylamine is ofthe formula:

wherein each R′ is independently hydrogen or methyl; R is naphthyl or

R″ and R′″ are halo, trifluoromethyl, C₁-C₄ alkyl, C₁-C₃ alkoxy or C₃-C₄alkenyl; and n and m are 0, 1 or 2; and acid addition salts thereofformed with pharmaceutically acceptable acids.
 15. The method of claim10, wherein said at least one 3-aryloxy-3-phenylpropylamine is selectedfrom the group consisting of 3-(p-isopropoxyphenoxy)-3-phenylpropylaminemethanesulfonate, N,N-dimethyl3-(3′,4′-dimethoxyphenoxy)-3-phenylpropylamine p-hydroxybenzoate,N,N-dimethyl 3-(alpha-naphthoxy)-3-phenylpropylamine bromide,N,N-dimethyl 3-(beta-naphthoxy)-3-phenyl-1-methylpropylamine iodide,3-(2′-methyl-4′,5′-dichlorophenoxy)-3-phenylpropylamine nitrate,3-(p-t-butylphenoxy)-3-phenylpropylamine glutarate, N-methyl3-(2′-chloro-p-tolyloxy)-3-phenyl-1-methylpropylamine lactate,3-(2′,4′-dichlorophenoxy)-3-phenyl-2-methylpropylamine citrate,N,N-dimethyl 3-(m-anisyloxy)-3-phenyl-1-methylpropylamine maleate,N-methyl 3-(p-tolyloxy)-3-phenylpropylamine sulfate, N,N-dimethyl3-(2′,4′-difluorophenoxy)-3-phenylpropylamine 2,4-dinitrobenzoate,3-(o-ethylphenoxy)-3-phenylpropylamine dihydrogen phosphate,N-methyl-(2′-chloro-4′-isopropylphenoxy)-3-phenyl-2-methylpropylaminemaleate, N,N-dimethyl 3-(2′-alkyl-4′-fluorophenoxy)-3-phenylpropylaminesuccinate, N,N-dimethyl 3-(o-isopropoxyphenoxy)-3-phenyl-propylaminephenyl acetate, N,N-dimethyl 3-(o-)bromophenoxy)-3-phenyl-propylaminebeta-phenylpropionate, N-methyl 3-(p-iodophenoxy)-3-phenyl-propylaminepropiolate, N-methyl 3-(3-n-propylphenoxy)-3-phenyl-propylaminedecanoate, and N-methyl3-(p-trifluoromethylphenoxy)-3-phenylpropylamine.
 16. The method ofclaim 10, wherein said at least one 3-aryloxy-3-phenylpropylamine isN-methyl 3-(p-trifluoromethylphenoxy) -3-phenylpropylamine or apharmaceutically acceptable salt thereof.
 17. The method of claim 10,wherein said at least one chemotherapeutic agent is selected from thegroup consisting of an alkylating agent, an antimetabolite, a naturalproduct, a miscellaneous agent, a hormone and an antagonist.
 18. Amethod of treating a subject suspected as having, or having a multidrugresistance cancer, the method comprising administering to the subject achemotherapeutically effective amount of at least one chemotherapeuticagent and a chemosensitizing effective amount of at least one3-aryloxy-3-phenylpropylamine, said chemosensitizing effective amountranges between about 0.1 mg/M² and about 10 mg/M².
 19. The method ofclaim 1 8, wherein said administering said at least one chemotherapeuticagent and said at least one 3-aryloxy-3-phenylpropylamine is performedsubstantially at the same.
 20. The method of claim 18, wherein saidcancer is selected from the group consisting of leukemia, lymphoma,carcinoma and sarcoma.
 21. The method of claim 18, wherein said at leastone 3-aryloxy-3-phenylpropylamine is administered orally.
 22. The methodof claim 18, wherein said at least one 3-aryloxy-3-phenylpropylamine isof the formula:

wherein each R′ is independently hydrogen or methyl; R is naphthyl or

R′ and R′″ are halo, trifluoromethyl, C₁-C₄ alkyl, C₁-C₃ alkoxy or C₃-C₄alkenyl; and n and m are 0, 1 or 2; and acid addition salts thereofformed with pharmaceutically acceptable acids.
 23. The method of claim18, wherein said at least one 3-aryloxy-3-phenylpropylamine is selectedfrom the group consisting of 3-(p-isopropoxyphenoxy)-3-phenylpropylaminemethanesulfonate, N,N-dimethyl3-(3′,4′-dimethoxyphenoxy)-3-phenylpropylamine p-hydroxybenzoate,N,N-dimethyl 3-(alpha-naphthoxy)-3-phenylpropylamine bromide,N,N-dimethyl 3-(beta-naphthoxy)-3-phenyl-1-methylpropylamine iodide,3-(2′-methyl-4′,5′-dichlorophenoxy)-3-phenylpropylamine nitrate,3-(p-t-butylphenoxy)-3-phenylpropylamine glutarate, N-methyl3-(2′-chloro-p-tolyloxy)-3-phenyl-1-methylpropylamine lactate,3-(2′,4′-dichlorophenoxy)-3-phenyl-2-methylpropylamine citrate,N,N-dimethyl 3-(m-anisyloxy)-3-phenyl-1-methylpropylamine maleate,N-methyl 3-(p-tolyloxy)-3-phenylpropylamine sulfate, N,N-dimethyl3-(2′,4′-difluorophenoxy)-3-phenylpropylamine 2 4-dinitrobenzoate,3-(o-ethylphenoxy)-3-phenylpropylamine dihydrogen phosphate,N-methyl-(2′-chloro-4′-isopropylphenoxy)-3-phenyl-2-methylpropylaminemaleate, N,N-dimethyl 3-(2′-alkyl-4′-fluorophenoxy)-3-phenylpropylaminesuccinate, N,N-dimethyl 3-(o-isopropoxyphenoxy)-3-phenyl-propylaminephenylacetate, N,N-dimethyl 3-(o-)bromophenoxy)-3-phenyl-propylaminebeta-phenylpropionate, N-methyl 3-(p-iodophenoxy)-3-phenyl-propylaminepropiolate, N-methyl 3-(3-n-propylphenoxy)-3-phenyl-propylaminedecanoate, and N-methyl3-(p-trifluoromethylphenoxy)-3-phenylpropylamine.
 24. The method ofclaim 18, wherein said at least one 3-aryloxy-3-phenylpropylamine isN-methyl 3-(p-trifluoromethylphenoxy) -3-phenylpropylamine or apharmaceutically acceptable salt thereof.
 25. The method of claim 18,wherein said at least one chemotherapeutic agent is selected from thegroup consisting of an alkylating agent, an antimetabolite, a naturalproduct, a miscellaneous agent, a hormone and an antagonist.
 26. Amethod of selecting a chemotherapeutic agent for which3-aryloxy-3-phenylpropylamine is a chemosensitizer comprising: assayingcytotoxicity of a candidate chemotherapeutic agent in the presence andin the absence of a 3-aryloxy-3-phenylpropylamine; and selecting acandidate chemotherapeutic agent as a chemotherapeutic agent for which3-aryloxy-3-phenylpropylamine is a chemosensitizer when the cytotoxicityof the candidate agent is greater in the presence of3-aryloxy-3-phenylpropylamine than in the absence of3-aryloxy-3-phenylpropylamine.
 27. The method of claim 26, wherein saidassaying is performed with multidrug resistant cells.
 28. The method ofclaim 26, wherein said assaying is performed using a3-aryloxy-3-phenylpropylamine at a dose that ranges between about 1 μMand about 10 μM.
 29. The method of claim 26, wherein when said assayingis performed in the presence of a 3-aryloxy-3-phenylpropylamine, said3-aryloxy-3-phenylpropylamine and said candidate chemotherapeutic agentare administered substantially at the same time.
 30. The method of claim26, wherein said 3-aryloxy-3-phenylpropylamine is of the formula:

wherein each R′ is independently hydrogen or methyl; R is naphthyl or

R′ and R′″ are halo, trifluoromethyl, C₁-C₄ alkyl, C₁-C₃ alkoxy or C₁-C₄alkenyl; and n and m are 0, 1 or 2; and acid addition salts thereofformed with pharmaceutically acceptable acids.
 31. The method of claim26, wherein said 3-aryloxy-3-phenylpropylamine is selected from thegroup consisting of 3-(p-isopropoxyphenxoy)-3-phenylpropylaminemethanesulfonate, N,N-dimethyl3-(3′,4′-dimethoxyphenoxy)-3-phenylpropylamine p-hydroxybenzoate,N,N-dimethyl 3-(alpha-naphthoxy)-3-phenylpropylamine bromide,N,N-dimethyl 3-(beta-naphthoxy)-3-phenyl-1-methylpropylamine iodide,3-(2′-methyl-4′,5′-dichlorophenoxy)-3-phenylpropylamine nitrate,3-(p-t-butylphenoxy)-3-phenylpropylamine glutarate, N-methyl3-(2′-chloro-p-tolyloxy)-3-phenyl-1-methylpropylamine lactate,3-(2′,4′-dichlorophenoxy)-3-phenyl-2-methylpropylamine citrate,N,N-dimethyl 3-(m-anisyloxy)-3-phenyl-1-methylpropylamine maleate,N-methyl 3-(p-tolyloxy)-3-phenylpropylamine sulfate, N,N-dimethyl3-(2′,4′-difluorophenoxy)-3-phenylpropylamine 2,4-dinitrobenzoate,3-(o-ethylphenoxy)-3-phenylpropylamine dihydrogen phosphate,N-methyl-(2′-chloro-4′-isopropylphenoxy)-3-phenyl-2-methylpropylaminemaleate, N,N-dimethyl 3-(2′-alkyl-4′-fluorophenoxy)-3-phenylpropylaminesuccinate, N,N-dimethyl 3-(o-isopropoxyphenoxy)-3-phenyl-propylaminephenylacetate, N,N-dimethyl 3-(o-)bromophenoxy)-3-phenyl-propylaminebeta-phenylpropionate, N-methyl 3-(p-iodophenoxy)-3-phenyl-propylaminepropiolate, N-methyl 3-(3-n-propylphenoxy)-3-phenyl-propylaminedecanoate, and N-methyl3-(p-trifluoromethylphenoxy)-3-phenylpropylamine.
 32. The method ofclaim 26, wherein said 3-aryloxy-3-phenylpropylamine is N-methyl3-(p-trifluoromethylphenoxy)-3-phenylpropylamine or a pharmaceuticallyacceptable salt thereof.
 33. The method of claim 26, wherein saidchemotherapeutic agent is selected from the group consisting of analkylating agent, an antimetabolite, a natural product, a miscellaneousagent, a hormone and an antagonist.
 34. A pharmaceutical compositioncomprising as a chemotherapeutically active ingredient at least onechemotherapeutic agent and as a chemosensitization active ingredient atleast one 3-aryloxy-3-phenylpropylamine.
 35. The pharmaceuticalcomposition of claim 34, packaged in a packaging material and identifiedin print in or on said packaging material, for use in the treatment of amultidrug resistance cancer.
 36. The pharmaceutical composition of claim34, wherein said at least one 3-aryloxy-3-phenylpropylamine is of theformula:

wherein each R′ is independently hydrogen or methyl; R is naphthyl or

R′ and R′″ are halo, trifluoromethyl, C₁-C₄ alkyl, C₁-C₃ alkoxy or C₃-C₄alkenyl; and n and m are 0, 1 or 2; and acid addition salts thereofformed with pharmaceutically acceptable acids.
 37. The pharmaceuticalcomposition of claim 34, wherein said at least one3-aryloxy-3-phenylpropylamine is selected from the group consisting of3-(p-isopropoxyphenxoy)-3-phenylpropylamine methanesulfonate,N,N-dimethyl 3-(3′,4′-dimethoxyphenoxy)-3-phenylpropylaminep-hydroxybenzoate, N,N-dimethyl 3-(alpha-naphthoxy)-3-phenylpropylaminebromide, N,N-dimethyl 3-(beta-naphthoxy)-3-phenyl-1-methylpropylamineiodide, 3-(2′-methyl-4′,5′-dichlorophenoxy)-3-phenylpropylamine nitrate,3-(p-t-butylphenoxy)-3-phenylpropylamine glutarate, N-methyl3-(2′-chloro-p-tolyloxy)-3-phenyl-1-methylpropylamine lactate,3-(2′,4′-dichlorophenoxy)-3-phenyl-2-methylpropylamine citrate,N,N-dimethyl 3-(m-anisyloxy)-3-phenyl-1-methylpropylamine maleate,N-methyl 3-(p-tolyloxy)-3-phenylpropylamine sulfate, N,N-dimethyl3-(2′,4′-difluorophenoxy)-3-phenylpropylamine 2,4-dinitrobenzoate,3-(o-ethylphenoxy)-3-phenylpropylamine dihydrogen phosphate,N-methyl-(2′-chloro-4′-isopropylphenoxy)-3-phenyl-2-methylpropylaminemaleate, N,N-dimethyl 3-(2′-alkyl-4′-fluorophenoxy)-3-phenylpropylaminesuccinate, N,N-dimethyl 3-(o-isopropoxyphenoxy)-3-phenyl-propylaminephenyl acetate, N,N-dimethyl 3-(o-)bromophenoxy)-3-phenyl-propylaminebeta-phenylpropionate, N-methyl 3-(p-iodophenoxy)-3-phenyl-propylaminepropiolate, N-methyl 3-(3-n-propylphenoxy)-3-phenyl-propylaminedecanoate, and N-methyl3-(p-trifluoromethylphenoxy)-3-phenylpropylamine.
 38. The pharmaceuticalcomposition of claim 34, wherein said at least one3-aryloxy-3-phenylpropylamine is N-methyl3-(p-trifluoromethylphenoxy)-3-phenylpropylamine or a pharmaceuticallyacceptable salt thereof.
 39. The pharmaceutical composition of claim 34,wherein said at least one chemotherapeutic agent is selected from thegroup consisting of an alkylating agent, an antimetabolite, a naturalproduct, a miscellaneous agent, a hormone and an antagonist.
 40. Apharmaceutical kit comprising as a chemotherapeutically activeingredient at least one chemotherapeutic agent and as achemosensitization active ingredient at least one3-aryloxy-3-phenylpropylamine, wherein said at least onechemotherapeutic agent and said at least one3-aryloxy-3-phenylpropylamine are individually packaged within thepharmaceutical kit.
 41. The pharmaceutical kit of claim 40, identifiedin print for use in the treatment of a multidrug resistance cancer. 42.The pharmaceutical kit of claim 40, wherein said at least one3-aryloxy-3-phenylpropylamine is of the formula:

wherein each R′ is independently hydrogen or methyl; R is naphthyl or

R′ and R′″ are halo, trifluoromethyl, C₁-C₄ alkyl, C₁-C₃ alkoxy or C₃-C₄alkenyl; and n and m are 0, 1 or 2; and acid addition salts thereofformed with pharmaceutically acceptable acids.
 43. The pharmaceuticalkit of claim 40, wherein said at least one 3-aryloxy-3-phenylpropylamineis selected from the group consisting of3-(p-isopropoxyphenxoy)-3-phenylpropylamine methanesulfonate,N,N-dimethyl 3-(3′,4′-dimethoxyphenoxy)-3-phenylpropylaminep-hydroxybenzoate, N,N-dimethyl 3-(alpha-naphthoxy)-3-phenylpropylaminebromide, N,N-dimethyl 3-(beta-naphthoxy)-3-phenyl-1-methylpropylamineiodide, 3-(2′-methyl-4′,5′-dichlorophenoxy)-3-phenylpropylamine nitrate,3-(p-t-butylphenoxy)-3-phenylpropylamine glutarate, N-methyl3-(2′-chloro-p-tolyloxy)-3-phenyl-1-methylpropylamine lactate,3-(2′,4′-dichlorophenoxy)-3-phenyl-2-methylpropylamine citrate,N,N-dimethyl 3-(m-anisyloxy)-3-phenyl-1-methylpropylamine maleate,N-methyl 3-(p-tolyloxy)-3-phenylpropylamine sulfate, N,N-dimethyl3-(2′,4′-difluorophenoxy)-3-phenylpropylamine 2,4-dinitrobenzoate,3-(o-ethylphenoxy)-3-phenylpropylamine dihydrogen phosphate,N-methyl-(2′-chloro-4′-isopropylphenoxy)-3-phenyl-2-methylpropylaminemaleate, N,N-dimethyl 3-(2′-alkyl-4′-fluorophenoxy)-3-phenylpropylaminesuccinate, N,N-dimethyl 3-(o-isopropoxyphenoxy)-3-phenyl-propylaminephenylacetate, N,N-dimethyl 3-(o-)bromophenoxy)-3-phenyl-propylaminebeta-phenylpropionate, N-methyl 3-(p-iodophenoxy)-3-phenyl-propylaminepropiolate, N-methyl 3-(3-n-propylphenoxy)-3-phenyl-propylaminedecanoate, and N-methyl3-(p-trifluoromethylphenoxy)-3-phenylpropylamine.
 44. The pharmaceuticalkit of claim 40, wherein said at least one 3-aryloxy-3-phenylpropylamineis N-methyl 3-(p-trifluoromethylphenoxy) -3-phenylpropylamine or apharmaceutically acceptable salt thereof.
 45. The pharmaceutical kit ofclaim 40, wherein said at least one chemotherapeutic agent is selectedfrom the group consisting of an alkylating agent, an antimetabolite, anatural product, a miscellaneous agent, a hormone and an antagonist. 46.A pharmaceutical composition comprising as an active ingredient at leastone 3-aryloxy-3-phenylpropylamine the pharmaceutical composition beingpackaged and indicated for use in chemosensitization, in combinationwith a chemotherapeutic agent and/or in a medical condition for whichchemosensitization is beneficial.
 47. The pharmaceutical composition ofclaim 46, wherein said at least one 3-aryloxy-3-phenylpropylamine is ofthe formula:

wherein each R′ is independently hydrogen or methyl; R is naphthyl or

R′ and R′″ are halo, trifluoromethyl, C₁-C₄ alkyl, C₁-C₃ alkoxy or C₃-C₄alkenyl; and n and m are 0, 1 or 2; and acid addition salts thereofformed with pharmaceutically acceptable acids.
 48. The pharmaceuticalcomposition of claim 46, wherein said at least one3-aryloxy-3-phenylpropylamine is selected from the group consisting of:3-(p-isopropoxyphenxoy)-3-phenylpropylamine methanesulfonate,N,N-dimethyl 3-(3′,4′-dimethoxyphenoxy)-3-phenylpropylaminep-hydroxybenzoate, N,N-dimethyl 3-(alpha-naphthoxy)-3-phenylpropylaminebromide, N,N-dimethyl 3-(beta-naphthoxy)-3-phenyl-1-methylpropylamineiodide, 3-(2′-methyl-4′,5′-dichlorophenoxy)-3-phenylpropylamine nitrate,3-(p-t-butylphenoxy)-3-phenylpropylamine glutarate, N-methyl3-(2′-chloro-p-tolyloxy)-3-phenyl-1-methylpropylamine lactate,3-(2′,4′-dichlorophenoxy)-3-phenyl-2-methylpropylamine citrate,N,N-dimethyl 3-( n-anisyloxy)-3-phenyl-1-methylpropylamine maleate,N-methyl 3-(p-tolyloxy)-3-phenylpropylamine sulfate, N,N-dimethyl 3-(2′,4′-difluorophenoxy)-3-phenylpropylamine 2,4-dinitrobenzoate,3-(o-ethylphenoxy)-3-phenylpropylamine dihydrogen phosphate, N-methyl-(2′-chloro-4′-isopropyl phenoxy)-3-phenyl-2-methylpropylamine maleate,N,N-dimethyl 3-(2′-alkyl-4′-fluorophenoxy)-3-phenylpropylaminesuccinate, N,N-dimethyl 3-(o-isopropoxyphenoxy)-3-phenyl-propylaminephenylacetate, N,N-dimethyl 3-(o-)bromophenoxy)-3-phenyl-propylaminebeta-phenylpropionate, N-methyl 3-(p-iodophenoxy)-3-phenyl-propylaminepropiolate, N-methyl 3-(3-n-propylphenoxy)-3-phenyl-propylaminedecanoate, and N-methyl3-(p-trifluoromethylphenoxy)-3-phenylpropylamine.
 49. The pharmaceuticalcomposition of claim 46, wherein said at least one3-aryloxy-3-phenylpropylamine is N-methyl3-(p-trifluoromethylphenoxy)-3-phenylpropylamine or a pharmaceuticallyacceptable salt thereof.
 50. The pharmaceutical composition of claim 46,wherein said at least one chemotherapeutic agent is selected from thegroup consisting of an alkylating agent, an antimetabolite, a naturalproduct, a miscellaneous agent, a hormone and an antagonist.